British journal of pharmacology 2015 volume 172 part 8

Thus, despite several decades of research on precondi-tioning, postconditioning and pharmacological condi-tioning of the heart, we have yet to see therapeutic realization of the potentia

Themed Section: Conditioning the Heart – Pathways to Translation

EDITORIAL

‘Conditioning the heart’ –

lessons we have learned

from the past and future

perspectives for new and

old conditioning ‘drugs’

Nina C Weber, PhD

Department of Anaesthesiology, Laboratory of Experimental Intensive Care and Anaesthesiology

(L.E.I.C.A) Academic Medical Centre (AMC), Meibergdreef 9, 1100 DD Amsterdam,

The Netherlands

Correspondence

Nina C Weber, PhD, AcademicMedical Centre (AMC),Department of AnaesthesiologyUniversity of Amsterdam,Laboratory of Experimental andClinical Experimental

Anaesthesiology (L.E.I.C.A.),Academic Medical Centre (AMC),Meibergdreef 9, 1100 DDAmsterdam, the Netherlands,

Almost a year ago my 6-year-old son asked me: ‘Mom, what is

it you are doing every day in your laboratory?’ First of all I

started explaining in children’s terms what the noble gas

helium is, what it can do, and why we think that it is not only

good to blow up balloons but might also be good for the heart

of some patients

However, he was not satisfied by this answer and

pro-ceeded asking things like: ‘ and why are you doing this?

What will you do next? How long have you been doing this?’

Finally, I ended up saying something that I had already

realized for quite a while: ‘Actually, I do not know why I am

still doing this “conditioning” research.’ After having said so,

I was quite upset as I have never been honest enough to

myself to admit that most of the research others and we do is

still not translated to the clinical situation Do we really help

patients to get better?

Thus, despite several decades of research on

precondi-tioning, postconditioning and pharmacological

condi-tioning of the heart, we have yet to see therapeutic

realization of the potential powerful protective effects of

conditioning on infarction, mechanical dysfunction and

arrhythmias associated with acute myocardial ischaemia

and reperfusion

Eventually, I was searching for supportive views to get back

on track with my own research, and I must admit that theoverwhelming participation of excellent researchers in thisthemed issue gave me a lot of motivation to hang on to thepathway of getting cardioprotective strategies by conditioningtranslated from the experimental laboratory to the patient.This is where the idea was born to bring together research-ers from all over the world to share their thoughts about thisquestion in the themed issue: ‘Conditioning the heart-Pathways to translation-scope for drug discovery’

Summary of content

Cohen and Downey are pioneers in the area of ‘conditioning’the heart and their review takes us through a brilliant histori-cal journey going back to 1971 when Maroko and co-workerssuggested that ST-segment shifts might be used as a markerfor infarct size reduction, and that infarct size reductionwould be a therapeutic option to prevent cardiac damage byischaemia/reperfusion (I/R) injury Four decades later, passing

pioneers like Hearse and Murry, Cohen et al review several

pathways of pre- and post-conditioning that have been cidated (Cohen & Downey, 2015) The endogenous mediators

elu-of the trigger phase include adenosine, bradykinin, opioids

BJP British Journal of

and other extracellular signalling molecules All these triggers

have been shown to bind to G-protein coupled receptors, but

surprisingly they then activate different pathways that

con-verge upon PKC Additionally, the mediator phase of

condi-tioning, including enzymes from the so ‘called’ reperfusion

injury salvage kinase (RISK) pathway, is discussed All

signal-ling finally seems to lead to inhibition of the mitochondrial

permeability transition pore (mPTP), which is currently

sug-gested to be the end-effector of ischaemic preconditioning

Several years after the discovery of pre-conditioning it

became clear that the obviously strong endogenous

protec-tive intervention had to occur before an ischaemic insult,

making the clinical application very difficult, as ischemia

often already has taken place, for example, in patients

pre-senting with an acute myocardial infarction This is when the

idea of protective pharamacological interventions during

rep-erfusion, possibly achieved by an intervention as ischaemic

post-conditioning, was born.

However, as Cohen and Downey (2015) point out,

irrespec-tive of the logical implications of the successful interventions

seen in animal experiments, several clincial trials using

ischae-mic miischae-micking agents like, adenosine, atrial natriuretic

peptide, or cyclosporine A, have yet remained little successful

In the last part of their review, Cohen and Downey focus

on the role of platelets, platelet activating factor (PAF) and

platelet P2Y2 inhibitors in cardioprotection (Cohen &

Downey, 2015) It is nowadays quite accepted that in patients

presenting with acute myocardial infarction (AMI),

compli-cations occurring during and after stenting can in fact be

significantly improved by the use of antiplatelet drugs like

aspirin, thienopyridines (clopidogrel and prasugrel) or

triazo-lopyrimidins (tricagrelor, Cohen & Downey, 2015) So far, the

anti-aggregation properties are suggested to be responsible for

the protective effect against re-occlusion of a stent, but

Cohen and Downey shed light upon a complete different

aspect They suggest that especially the P2Y2 ADP receptor

inhibitors (thienopyridines and triazolopyrimidines), in fact

(already) activate the post-conditioning pathway: This means

that the attempt to add another ‘conditioning’ stimulus by

e.g ischaemic or pharmacological conditioning might fail

(Cohen & Downey, 2015)

Cohen and Downey (2015) lead the reader to a very

critical but also future-oriented view on ‘conditioning’ of the

heart They support further laboratory research for

elucidat-ing alternative pathways that may extend the spectrum of the

anti-ischaemic armamentarium beyond the platelet

inhibi-tors used in standard care

Baxter and Bice take a closer look into the phenomenon

of ‘post-conditioning’ of the heart (Bice & Baxter, 2015) This

more recently described form of ‘conditioning’ has been in

the focus of research over the last decade Bice and Baxter

(2015) distinctively introduce the mechanisms that have

been implicated in ischaemic post-conditioning and which

are quite similar to those known from pre-conditioning

Again, inhibition of the mPTP resembles the potential

end-effector in post-conditioning and pathways like the RISK,

SAFE, GSK-3 beta and cGMP/PKG are involved (Bice & Baxter,

2015) Bice and Baxter (2015) come to the conclusion, that

even though promising results from initial clinical studies

using both, ischaemic and pharmacological

post-conditioning exist, here the final translation to the clinical

situation fails They suggest that study design, timing, drugadministration, technical limitations with respect to theend-point measurements and – most importantly – existingco-morbidities in our patients might limit the translation ofprotection by conditioning to the clinic (Bice & Baxter, 2015).However, as Cohen and Downey, they end with a very prom-ising view on the dead end some of the ‘conditioning’researchers might feel to be stranded in In their view thevariability within the group of AMI patient makes it probablyimpossible to find a one-size-fits-all cure for each patient, buttaking into account the enormous amount of patients withcardiovascular diseases, any standard drug in a single dosemight make a huge difference at least for some of the respec-tive patients (Bice & Baxter, 2015)

Another more clinically applicable form of ‘conditioning’

the heart, remote ischaemic conditioning (RIC), is described in the review of Schmidt et al (2015) RIC is defined as short-

lasting periods of ischaemia applied to a distant organ formthe heart, which eventually lead to the protection of theheart itself against ischaemia reperfusion injury Schmidt

et al draw a picture of the most recent and promising

media-tors and targets involved in remote ischaemic conditioning.Among these, especially the recently extensively investigateddialysate from plasma, containing most probably not one butseveral potent cardioprotective factors (factor X), is discussed.Also MicroRNA (especially miR-144) and exosome releaseduring RIC have recently entered the focus of RIC research

Schmidt et al suggest that these might be very promising

strategies to develop future therapies mimicking RIC

(Schmidt et al., 2015) In addition, Pzyklenk points out that

among all forms of ‘conditioning’ without doubt conditioning’ and RIC are the most promising strategies to betranslated to the clinic In her view, especially the extremelycomplex and time sensitive signalling network involving allconditioning forms limits translation from promising pre-clinical trials to larger clinical trials She also critically ques-tions the study design (patient population) of recent clinicaltrials examining conditioning the heart as well as the choice

‘post-of the experimental protocol (Przyklenk, 2015) In her review,the reader will be confronted with the hypothesis that despitethe assumption that translatability of preclinical data to theclinic would implicate a study design as close as possible tothe meanwhile well-established pathways of cardioprotec-tion, there is an enormous heterogeneity among and withinthe clinical studies that have been performed so far(Przyklenk, 2015)

The reviews of Pagliaro and Penna (2015) and Inserte andGarcia-Dorado (2015) deal with the complex mechanism

underlying reperfusion injury In this phase of I/R injury,

redox signalling (Pagliaro & Penna, 2015) and cGMP/PKGsignalling (Inserte & Garcia-Dorado, 2015) are criticallyinvolved in the development of cardiac damage Pagliaro andPenna (2015) point out to the fact that undifferentiateddiminishment of redox signalling [reactive oxygen species(ROS) and reactive nitrogen species (RNS)] by antioxidantscannot be the future therapy in I/R damage, as in fact redoxsignalling is vital to several physiological processes (Pagliaro

& Penna, 2015) The authors suggest a more site- and specific inhibition of ROS/RNS without affecting survivalpathways relying on ROS/RNS, however, clinical data areagain sparse regarding this topic (Pagliaro & Penna, 2015)

Next to ROS signalling, a pivotal role for cGMP/PKG

sig-nalling during reperfusion injury and cardioprotection by

‘conditioning’ has been described (Inserte & Garcia-Dorado,

2015) According to Inserte and Garcia-Dorado (2015)

exten-sive amounts of pre-clinical data definitively support the role

of cGMP/PKG in cardioprotection Moreover, the authors are

convinced that targeting these key players of the signal

trans-duction cascade in the early phase of reperfusion is a valuable

and very promising therapeutic option toward diminished

cardiac damage (Inserte & Garcia-Dorado, 2015)

Unfortu-nately, the narrow dose-response curve that has to be

fol-lowed when cGMP levels are increased might dampen the

positive view, as too high cGMP levels have recently been

shown to be rather harmful than protective (Inserte &

Garcia-Dorado, 2015)

The role of the extracellular signalling molecules

(auta-coids): adenosine, bradykinin and opioids, is extensively

described in the review by Kleinbongard and Heusch (2015)

and for opioids by Headrick et al (2015) These endogenous

signalling molecules are un-doubtfully all involved in the

different forms of ‘conditioning’ however, once again

transla-tions to clinical trials were disappointing Although e.g

adenosine has been tested in several trials of AMI, elective PCI

or CABG patients, and some studies are positive and

promis-ing, there is still no consensus on whether adenosine reduces

infarct size in the clinical scenario (Kleinbongard & Heusch,

2015) Headrick et al point out that although small clinical

trials showed a benefit of morphine and remifentanil in CABG

surgery patients, targeting the opioid receptors (OPR) more

specifically (e.g.δ-OPR agonists) in order to avoid

cardiorespi-ratory effects of unspecific OPR agonists would be a more

promising approach (Headrick et al., 2015) Especially for

opioids one has to take into account that maintaining

anaes-thesia during surgical procedures might in itself already be

cardioprotective and thus stocking up on cardioprotective

interventions might be difficult in this setting (Kleinbongard

& Heusch, 2015)

Both expert groups agree upon the fact that ageing,

co-morbidities and – most importantly – relevant drugs

during the surgical procedure are the challenge that has to be

overcome before autacoid mimicking drugs can find their

way into the clinic (Kleinbongard & Heusch, 2015) (Headrick

et al., 2015) In this context, sustained ligand-activated

pro-tection (SLP) might have a future role in clinical applications

as it has been shown to be effective also in diseased animal

models (Headrick et al., 2015) Kleinbongard and Heusch

(2015) end with a conclusion that might frustrate those of us

working on ‘pharmacological’ induced conditioning The

authors argue that probably the search for more ‘drugs’ to

induce cardioprotection should stop soon, and the

develop-ment of more reliable RIC models in the clinic could be the

future (Kleinbongard & Heusch, 2015)

The two reviews dealing with the probable most easily

translatable conditioning strategies using anaesthetics or

noble gases extensively describe the mechanisms underlying

such cardioprotection (Kikuchi et al., 2015, Smit et al., 2015).

Protection induced by volatile anaesthetics (Kikuchi et al.,

2015) and later on noble gases, like xenon and helium (Smit

et al., 2015), has been recognized for the last two decades.

Unfortunately, these two reviews come to the disappointing

conclusion that the application of these substances, although

already clinically used, is still limited and advances in this

field are minimal (Kikuchi et al., 2015, Smit et al., 2015).

Ageing, diabetes, hyperglycaemia and drugs frequently used

in AMI patients (beta blockers, glibenclamide) have beenshown to diminish this form of conditioning in animals as

well as in humans (Kikuchi et al., 2015) Thus once again,

more laboratory research using adapted animal models andmodels that properly mimic the clinical anaesthesia models

are needed (Kikuchi et al., 2015, Smit et al., 2015).

In the last four reviews some of the key targets involved inorchestrating different conditioning forms are excellently

reviewed (Ong et al., 2015, Halestrap et al., 2015, Martin

et al., 2015, Schilling et al., 2015) For the sake of brevity I will

only be able to highlight some snap shots from these reviews,hereby emphasizing that under no circumstances do I wish toundermine the importance of these contributions to thecompletion of this themed issue

Regarding the aforementioned crucial involvement ofmPTP in cardioprotection by different conditioning forms, the

reviews by Ong et al (2015) and by Halestrap et al (2015) give

an excellent detailed overview on the mechanisms by whichthe mPTP is regulated and in which way hexokinase 2 (HK2)might be of importance for maintenance of the opening of thispore The opening of the mPTP at the onset of reperfusionleads to cell death Trials over the past years using cyclosporine

A, an inhibitor of mPTP opening, have proven that whenadministered right at the beginning of reperfusion, it in fact

reduces MI (Ong et al., 2015) Larger multicentre trials as the

CYCLE and CIRUS study are currently running and will revealvery important results regarding the clinical use of cyclo-

sporine A (Ong et al., 2015).

The glycolytic enzyme HK2 has been found to bind to theouter membrane of the mitochondria, thereby stabilizing thecontact sites of outer and inner mitochondrial membranes.Dissociation of HK2 from the membrane during the ischae-mic phase leads to an increased loss of cytochrome C fromthe mitochondria, which in turn results in opening of the

mPTP during reperfusion (Halestrap et al., 2015) Ischaemic

preconditioning has been shown to involve increased HK2binding to the mitochondrial membrane, thereby inducing

cardioprotection Halstrap et al critically evaluate the

poten-tial development of drugs that might increase binding of HK2

to the mitochondria, as these are yet unavailable (Halestrap

et al., 2015).

Also caveolins, reviewed by Schilling et al (2015), have

more recently been associated with the mitochondria and ithas been convincingly shown that they play a pivotal role indifferent forms of conditioning Caveolins are structural pro-teins that are essential for the formation of so called ‘cave-olae’, small plasma membrane invaginations enriched with

cholesterol and sphingolipids (Schilling et al., 2015) These

proteins are thought to regulate protective signalling within amultiprotein (signalosome) complex Interestingly, many ofthe above-discussed key players (e.g opioids, adenosine,PKC) of ischaemic and pharmacological conditioning havebeen shown to be associated with caveolae/caveolins in a

dynamic process Schilling et al distinctively focus on the

fact that caveolins might be key players in overcoming thepathophysiological limits for conditioning (ageing, diabetes),hereby leaving us with the hope that future studies mightidentify drugs that can specifically increase caveolin expres-

BJP

‘Conditioning the heart’

sion, thereby protecting the heart against ischaemia/

reperfusion damage (Schilling et al., 2015).

Last but not least, Martin et al provide us with an

excel-lent review over the role of a member of the

‘stress-activated’ kinases family, the p38 MAPK in cardiovascular

disease The activation of p38 during ischaemic

precondi-tioning cycle has been shown to attenuate the detrimental

activation of the same enzyme during the lethal ischaemic

period Thus, p38 MAPK activation also might trigger

pro-tective effects in the heart, thereby making the inhibition of

p38 MAPK quite unpredictable in clinical trials (Martin

et al., 2015) However, very recent clinical trials with the

selective, potent, and orally active p38 MAPK inhibitor

Los-mapimod (GlaxoSmithKline, Brentford, London, UK) show

promising results in the settings of myocardial infarction,

and a much larger trial implementing Losmapimod is on its

way (Martin et al., 2015).

Concluding remarks

This themed issue summarizes a very important portion of all

the research ongoing in the area of cardioprotection by

several ‘conditioning’ forms Of course, it cannot be complete

as there is still so much to learn The main goal of this

themed issue was not only to review the current state of the

art in this field, but also to provide a critical overview upon the

opportunities that lie ahead for strong endogenous

mecha-nisms of conditioning to gain broader translatability into the

clinical situation

With regard to this, we hope that studying these review

articles will convince the readership that there is indeed a

future for ‘conditioning the heart’ against ischaemic damage

By combining the enormous amount of knowledge we have

gained from animal and preclinical studies, and applying this

knowledge to the existing co-morbidities and peculiarities

occurring during the perioperative period, we will hopefully

succeed in our venture/conquest to identify novel candidate

drugs for conditioning the heart and for testing in clinical

trials

References

Bice JS, Baxter GF (2015) Postconditioning signalling in the heart:

mechanisms and translatability Br J Pharmacol 172: 1933–1946

Cohen MV, Downey JM (2015) Signalling pathways andmechanisms of protection in pre- and postconditioning: historicalperspective and lessons for the future Br J Pharmacol 172:

1913–1932

Halestrap AP, Pereira GC, Pasdois P (2015) The role of hexokinase

in cardioprotection – mechanism and potential for translation Br JPharmacol 172: 2085–2100

Headrick JP, See Hoe LE, Du Toit EF, Peart JN (2014) Opioidreceptors and cardioprotection – ‘opioidergic conditioning’ of theheart Br J Pharmacol 172: 2026–2050

Inserte J, Garcia-Dorado D (2015) cGMP/PKG pathway as acommon mediator of cardioprotection Translatability andmechanism Br J Pharmacol 172: 1996–2009

Kikuchi C, Dosenovic S, Bienengraeber M (2015) Anaesthetics ascardioprotectants – translatability and mechanism Br J Pharmacol172: 2051–2061

Kleinbongard P, Heusch G (2015) Extracellular signalling molecules

in the ischaemic/reperfused heart – druggable and translatable forcardioprotection? Br J Pharmacol 172: 2010–2025

Martin ED, Bassi R, Marber MS (2015) p38 MAPK incardioprotection – are we there yet? Br J Pharmacol 172:

2101–2113

Ong S, Dongworth RK, Cabrera-Fuentes HA, Hausenloy DJ (2015).Role of the MPTP in conditioning the heart – translatability andmechanism Br J Pharmacol 172: 2074–2084

Pagliaro P, Penna C (2015) Redox signaling and cardioprotection –translatability and mechanism Br J Pharmacol 172: 1974–1995.Przyklenk K (2015) Ischemic conditioning: pitfalls on the path toclinical translation Br J Pharmacol 172: 1961–1973

Schilling JM, Roth DM, Patel HH (2015) Caveolins incardioprotection – translatability and mechanism Br J Pharmacol172: 2114–2125

Schmidt MR, Redington A, Botker HE (2015) Remote conditioningthe heart overview: translatability and mechanism Br J Pharmacol172: 1947–1960

Smit KF, Weber NC, Hollmann MW, Preckel B (2015) Noble gases

as cardio-protectants – translatability and mechanism Br JPharmacol 172: 2062–2073

Themed Section: Conditioning the Heart – Pathways to Translation

REVIEW

Signalling pathways and

mechanisms of protection in

pre- and postconditioning:

historical perspective and

lessons for the future

Michael V Cohen1,2and James M Downey1

1Departments of Physiology and2Medicine, University of South Alabama College of Medicine,

Mobile, AL, USA

mcohen@southalabama.edu -

to the hospital and being treated with platelet P2Y12receptor antagonists, the current standard of care, are indeed alreadybenefiting from protection from the conditioning pathways outlined earlier If that proves to be the case, then future attempts

to further decrease infarction will have to rely on interventions which protect by a different mechanism

exchanger; PAF, platelet-activating factor; PCI, percutaneous coronary intervention; PDK, 3

′-phosphoinositide-dependent kinase; PTCA, percutaneous transluminal coronary angioplasty; RISK, reperfusion injury survival kinases;ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; SAFE, survivor activating factor enhancement; SPHK1,sphingosine kinase; Src, sarcoma; STEMI, ST-segment-elevation myocardial infarction; TRAF-2, TNF receptor-associatedfactor 2

BJP British Journal of

With the exception of revascularization, ischaemic

precondi-tioning (IPC) is undeniably the most powerful

cardioprotec-tive intervention targeting ischaemia/reperfusion injury yet

to be identified All scientists agree that this intervention can

salvage ischaemic myocardium following a period of

ischae-mia and reperfusion and reduce infarct size, the original

observation and time-honored parameter of

cardioprotec-tion Yet this success in the experimental laboratory has yet to

be translated into a clinical procedure that has produced

equally satisfying results Thus, both scientists and clinicians

continue to search for the miraculous intervention that can

be applied to the patient with an acute myocardial infarction

(AMI) to decrease infarct size and diminish the clinical

seque-lae of coronary artery occlusion and reperfusion The closest

we have come to this is revascularization therapy In patients

with coronary occlusion and AMI, current standards demand

that the coronary artery be opened to reperfuse the ischaemic

myocardium Although tissue salvage understandably is

dependent on reflow, this revascularization paradoxically

creates injury of its own, so-called ‘reperfusion injury’ It is

the latter which most recent cardioprotective interventions

purport to target To appreciate why identification of an

appropriate cardioprotective agent has largely failed to date

and to provide hope that we may be close to developing an

effective approach, it is necessary to understand the historyand science of cardioprotection

Pioneering studies of infarct size modification

ST-segment shifts as a marker of infarct size

In 1971, Maroko, working in Braunwald’s laboratory, posed strategies for limiting necrosis following acute coro-

pro-nary occlusion (Maroko et al., 1971) Maroko et al recognized

the importance of infarct size on outcomes following tion and were the first to suggest that infarction might betherapeutically reduced These investigators mapped thedegree of ST-segment shifts on the anterior myocardialsurface at the end of 10-min coronary occlusions in dogs.After the heart was reperfused and had recovered, the occlu-sion and mapping were repeated after an experimental inter-vention The sum of the ST-segment shifts was thought toreflect the severity of the ischaemia If the sum of ST-segmentshifts was attenuated, it was concluded that the interventionhad protected the heart The concept was brilliant in that itallowed each animal to serve as its own control, but it relied

infarc-on the assumptiinfarc-on that the ST-segment sum represented trueinfarct size which, unfortunately, turned out to not always be

the case Maroko et al as well as subsequent investigators

Tables of Links

TARGETS

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

were also greatly handicapped by a lack of scientific

informa-tion as to how ischaemia/reperfusion actually kills heart

tissue The Braunwald laboratory focused on a supply–

demand relationship Demand could be reduced by

β-blockers and supply could be increased by interventions

thought to promote oxygen delivery such as hyaluronidase

(Maroko et al., 1972) As it turned out, the supply–demand

relationship was but only one determinant of cell death Yet

their pioneering efforts started a field of research that still

thrives today

Reperfusion injury: a paradox

Hearse et al (1973) introduced the ‘oxygen paradox’

Perfu-sion of a rat heart with hypoxic buffer for a prolonged period

seemed to have little consequence, but switching back to

oxygenated perfusate caused immediate cell destruction

While the reintroduction of oxygen was needed for recovery,

at the same time it was associated with an injury That was

the paradox The concept of reperfusion injury was very

attractive because at that time it was recognized that AMI was

caused by a coronary thrombus that could be dissolved with

a thrombolytic agent If much of the injury occurred at

rep-erfusion, it would not be too late to prevent it with some

intervention despite presentation of the patients with

ischae-mia in progress It was hypothesized that reintroduction of

oxygen produced a burst of free radicals that in turn led to

membrane damage, interference with ion pumps and volume

dysregulation A closely associated hypothesis was that

leu-kocytes would invade reperfused tissue and attack viable

myocytes by releasing free radicals Personnel in Lucchesi’s

laboratory concentrated on the role of free radicals in

myo-cardial infarction (Jolly et al., 1984) Thus, free radical

scav-engers appeared to decrease infarction in a canine model of

ischaemia/reperfusion Although these studies were

champi-oned by local advocates, the inconsistent results obtained in

other independent laboratories suggested problems with this

approach (Reimer et al., 1989) The same held for

investiga-tions of anti-inflammatory agents (Tissier et al., 2007a) The

reason for divergent results among the many studies of

anti-oxidant and anti-inflammatory agents have never been

resolved, but even in the most supportive studies salvage was

hardly greater than 10%, probably too modest to have

mean-ingful clinical impact One began to wonder if it was even

possible to alter the vulnerability of ischaemic myocardium

to infarction

IPC

Then, in 1986, Charles Murry, in the laboratory of Reimer

and Jennings, made a seminal observation (Murry et al.,

1986) It was reported that preceding a 40 min coronary

occlusion in dogs with four cycles of 5 min coronary

occlu-sion/5 min reperfusion would decrease the amount of

infarc-tion of the risk area subtended by the occluded vessel from 28

to 7% That was a 75% reduction in infarct size despite the

fact that those hearts endured an additional 20 min of

ischae-mia They called this phenomenon IPC Perhaps because of

Murry’s frankly antithetical observation that more ischaemia

was better, confirmation of the observation was not

immedi-ate Three years passed before scientific papers dealing with

IPC began to appear But when they did, confirmation was

overwhelming Those studies noted that the intervention

uniformly protected canine (Murry et al., 1986; Gross and

Auchampach, 1992), rodent (Liu and Downey, 1992; Yellon

et al., 1992), porcine (Schott et al., 1990), rabbit (Van Winkle

et al., 1991; Toombs et al., 1993), primate (Yang et al., 2010)

and even avian (Rischard and McKean, 1998) hearts frommyocardial infarction At last there was conclusive proof thatinfarct size could be modified, at least by this singular inter-vention of IPC Of course, therapeutic IPC of a heart would beimpossible to implement clinically in any setting except,perhaps, open heart surgery Translation of IPC into clinicalpractice would have to wait until its mechanism was betterunderstood before a treatment could be identified that could

be administered after ischaemia had begun

Mechanism of IPC: triggering phase

Surface receptors trigger IPC

The first insight into IPC’s mechanism was reported by Liu

et al (1991) They announced that IPC is triggered by

recep-tor occupancy Activation of the Gi-coupled adenosine A1

receptor in rabbits triggered IPC’s protection Thus, an sine receptor antagonist blocked IPC’s protection, while infu-sion of adenosine or an A1-selective agonist in lieu of brief

adeno-ischaemia duplicated IPC’s protection Liu et al proposed

that net dephosphorylation of ATP during ischaemia results

in production and release of adenosine which then wouldbind to A1adenosine receptors leading to a preconditionedphenotype So had these investigators defined IPC’s mecha-nism and were they ready to propose an intervention thatcould be used clinically? Hardly They had identified a phar-macological trigger, but unfortunately the trigger, like IPC,had to be given prior to the onset of ischaemia Identification

of more parts of IPC’s signal transduction pathway and of theoverall mechanism would be required

Opioid and bradykinin’s signalling

Two other endogenously released trigger substances,

brady-kinin (Wall et al., 1994) and opioids (Schultz et al., 1995),

were also found to be involved in IPC’s protective action.Inhibition of any of these three receptors aborted protectionfrom a single preconditioning cycle However, simply increas-ing the number of preconditioning cycles could restore pro-tection suggesting that the three receptors had an additiveeffect which was required to reach a protective threshold

(Goto et al., 1995) Thus, the additional cycles of ischaemia/

reperfusion produced increased stimulation of the two hibited receptors so that the protective threshold couldfinally be reached

unin-All three of these triggers, adenosine, bradykinin andopioids, bind to Gi-coupled receptors The proposed multipletrigger theory implies that all triggers converge on a common

target Ytrehus et al (1994) reported that PKC seemed to play

a major role in IPC and it was found that protection triggered

by any of the three receptors could be blocked by PKC

inhibi-tors (Goto et al., 1995; Sakamoto et al., 1995; Baines et al., 1997; Miki et al., 1998a) Thus, PKC is believed to be this

common target Hence, adenosine, bradykinin and opioidsbind to their respective receptors and the second messenger

BJP

Pre- and postconditioning signalling

G-protein is cleaved into activeα and βγ subunits which then

result in activation of PKC It would seem intuitive that the

various agonists coupled to common Gi-proteins should

trigger identical signalling However, mysteriously this is not

the case Adenosine, bradykinin and opioids activate very

divergent pathways; however, all three pathways eventually

converge on PKC

Opioid cardioprotection is dependent on downstream

metalloproteinase and EGF receptor (Cohen et al., 2007a).

This part of the signalling pathway was first mapped by

studying ACh-stimulated receptors, Gi-coupled receptors,

whose downstream protection is governed by signalling

similar to that of opioids (Krieg et al., 2002; 2004; Oldenburg

et al., 2002; 2003) While ACh is a potent trigger of

precon-ditioning’s protection, it is not a physiological trigger as

transient ischaemia does not cause its release ACh binds to

its receptor resulting in cleavage of Gi and subsequent

metalloproteinase-dependent cleavage of heparin-binding

EGF-like growth factor (HB-EGF) from membrane-associated

pro-HB-EGF (Figure 1) The liberated HB-EGF then activates

membrane-bound EGFR by binding to its ectodomain

result-ing in EGFR dimerization which in turn leads to

autophos-phorylation of tyrosine residues on both EGFRs and binding

of sarcoma (src) tyrosine kinase to form a signalling module.The latter attracts and activates PI3K

Bradykinin’s signalling is comparable, although a

differ-ent metalloproteinase is involved Methner et al (2009)

dem-onstrated involvement of metalloproteinase-8 and the EGFR.Downstream steps are similar to those for ACh and opioids

(Cohen et al., 2007a).

NO

PI3K-produced phosphorylated lipid metabolites dylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate induce Akt to translocate to the plasma

phosphati-membrane (Andjelkovic´ et al., 1997) where it is ylated by PDK1 and 2 (Stephens et al., 1998) and this initiates

phosphor-a signphosphor-alling cphosphor-ascphosphor-ade Akt phosphor-activphosphor-ates ERK phosphor-and endotheliphosphor-al NOS

(Dimmeler et al., 1999) The latter enzyme catalyzes

produc-tion of NO which stimulates guanylyl cyclase (GC) GC lyzes the production of cGMP which itself activates PKG(Figure 1)

cata-NO is a gaseous free radical and important biological

regulator and cellular signalling molecule In 1992, Vegh et al.

(1992) proposed that endogenous NO might be involved inpreconditioning This announcement triggered a significant

Figure 1

controversy regarding the precise role of NO in IPC

(Weselcouch et al., 1995) to which we unwittingly

contrib-uted In a study in in vitro rabbit hearts published in 2000,

we noted that Nω-nitro-L-arginine methyl ester (L-NAME), a

NOS inhibitor, had no effect on the dramatic protection

induced by IPC, whereas the NO donor

S-nitroso-N-acetylpenicillamine administered before the index ischaemia

in lieu of the repeated brief 5 min coronary occlusions

mim-icked IPC and protected hearts (Nakano et al., 2000) We

concluded that exogenously administered NO could trigger

the preconditioned state, but that endogenous production of

NO was not involved in IPC This conundrum was not

resolved for several years until we repeated studies with

L-NAME in IPC in in vivo rabbit hearts (Cohen et al., 2006).

L-NAME blocked the protection of IPC Our earlier

observa-tions, although accurate, were dependent on the in vitro

model used As seen in Figure 1, bradykinin, opioids and

adenosine are released by the ischaemic heart But in the

isolated, buffer-perfused heart, the absence of circulating

kininogens would minimize release of bradykinin In

addi-tion, opioid release would be attenuated because of the

absence of cardiac innervation Therefore, virtually all

trig-gering would be the result of adenosine release which

bypasses the NO-dependent trigger pathway (Figure 1)

As noted earlier, classical signalling dogma indicates that

NO stimulates GC leading to generation of cGMP which in

turn activates PKG (Figure 1) Studies with activators and

inhibitors of PKG and cGMP analogues (Han et al., 2002;

Oldenburg et al., 2004; Qin et al., 2004; Kuno et al., 2008)

clearly demonstrated the involvement of PKG in IPC, and

Baxter’s laboratory (D’Souza et al., 2003; Burley et al., 2007)

demonstrated increased myocardial levels of cGMP after

pro-tection by B-type natriuretic peptide Additionally, BAY

58-2667, a NO-independent GC activator, conditioned rat

and rabbit hearts (Krieg et al., 2009) Thus, in addition to

proven involvement of endogenous NO, there is much

evi-dence to support participation of GC and PKG in

condition-ing’s protection

Several investigators have also demonstrated involvement

of a NO-mediated, PKG-independent signalling pathway (Sun

et al., 2013; Penna et al., 2014) NO can directly modify

sulf-hydryl residues by S-nitrosylation The latter is an important

post-translational protein modification in signalling IPC

increases S-nitrosylation and IPC cardioprotection can be

aborted by treatment with ascorbate which is a reducing

agent resulting in specific degradation of S-nitrosylated

com-pounds Additionally, in isolated mouse hearts,

pharmaco-logic inhibition of the soluble GC/cGMP/PKG pathway failed

to block IPC-induced cardioprotection Thus, in at least some

models, this alternative pathway of NO signalling may be

important and it is possible that each pathway may

contrib-ute to cardioprotection and the ischaemic stimulus itself or

other unidentified factors may determine whether one or the

other pathway is utilized Once again, there is redundancy to

the response to ischaemia which may be an adaptive change

insuring a maximal cardioprotective result

ATP-sensitive potassium channels and

redox signalling

The next critical step in this signalling cascade is opening of

an ATP-sensitive K+channel (KATP) At the same time that we

were uncovering the importance of adenosine in IPC, GarretGross’ laboratory was doing studies with KATPchannels Thoseinvestigators found that glibenclamide, a blocker of thechannel, could also selectively block IPC’s protection (Grossand Auchampach, 1992) Similarly, pretreatment with a KATP

channel opener mimicked the protection (Gross andAuchampach, 1992) For a while it seemed that it had to beeither adenosine or potassium channels which governed pro-tection, but it turned out that they were simply two links inthe same chain; both were involved Several subsequentstudies revealed that the KATPchannel in question was locatednot on the sarcolemma but in the inner mitochondrial mem-

brane (Garlid et al., 1997; Liu et al., 1998).

Mitochondrial KATPchannels (mtKATP) are not triggers forIPC but rather are a critical link in the signalling pathway

between surface receptors and PKC (Pain et al., 2000).

Opening of mtKATP is PKG-dependent (Costa et al., 2005;

2008), but the channels are obviously not accessible to solic PKG There are intermediate steps involving PKCε in themitochondria which transmits the signal from cytosolic PKG

cyto-to the mtKATPchannel (Costa et al., 2005; 2008; Jabu˚rek et al.,

2006) One theory proposes that PKG reaches the dria via signalosomes that bud off of sarcolemmal caveolae

mitochon-and contain critical signalling enzymes (Garlid et al., 2009).

Channel opening permits K+ to enter the matrix along itselectrochemical gradient K+ influx is balanced by electro-genic H+efflux driven by the respiratory chain

An important link in this signalling is the redox coupling

of mtKATPchannel opening and PKC activation Forbes et al.

(2001) were the first to recognize this link when they noticedthat either of the antioxidants N-acetylcysteine or N-2-mercaptopropionylglycine could block the protection fromthe mtKATPopener, diazoxide It is not known exactly howmtKATPopening causes production of free radicals, but onetheory is that mtKATP-dependent matrix alkalinization affectscomplex I and/or III which are poised to generate increasedamounts of superoxide and its products H2O2and hydroxylradical (Costa and Garlid, 2008) All of the signalling steps tothis point occur in ischaemic cells However, generation ofthis burst of reactive oxygen species (ROS) must await rein-troduction of oxygen into the myocardium which occursduring the reflow phase of the preconditioning cycle ofischaemia/reflow There are many PKC isozymes It appearsthat activation of PKCε is necessary and sufficient to achievecardioprotection, while activation of PKCδ specifically blocks

protection (Dorn et al., 1999; Ping et al., 2002; Inagaki et al.,

2003a,b) Thus, activation of PKC continues the signallingcascade

The relationship among mtKATP, ROS and PKC is poorlyunderstood While ROS can directly activate PKC by causingrelease of Zn++from the regulatory domain (Korichneva et al.,

2002), connexin 43 (Cx43) appears to be a vital link in redoxsignalling Cx43 which makes most of the gap junctionsbetween cardiomyocytes was noted to be necessary for pre-

conditioning’s protection (Schwanke et al., 2002) It was later

noted that protection depended on a mitochondrial tion of Cx43 hemichannels located on the inner membrane.Depletion of these channels attenuates both protection andROS production from an mtKATPopener (Heinzel et al., 2005).

popula-Most recently, it was shown that an mtKATP opener causesphosphorylation of Cx43 by PKC and that phosphorylation is

BJP

Pre- and postconditioning signalling

required for protection (Srisakuldee et al., 2009) This suggests

some sort of circular signalling circuit as phosphoCx43 is

needed for ROS production and ROS cause PKC activation,

but PKC phosphorylates Cx43 The role actually played by

Cx43 in the protective process (e.g a channel, a signalling

molecule or a scaffold) is still a mystery

The redox signalling step explains one of the mysteries of

IPC Why is the heart protected when a prolonged ischaemic

period is preceded by a short coronary occlusion followed by

reperfusion but yet is not protected during a single prolonged

insult? All of the trigger receptors are activated during the

single prolonged ischaemic insult, but signalling stops at the

step requiring redox coupling to PKC because of the lack of

oxygen which is supplied during IPC’s short reperfusion A

ROS scavenger blocks protection from IPC and it can easily be

seen that the critical time for that blockade is during IPC’s

reperfusion phase (Dost et al., 2008) While ROS-sensitive

dyes indicate that radical production can occur during

ischae-mia (Becker et al., 1999), apparently the ROS species

gener-ated is not one capable of the redox signalling We also have

found that reperfusing with hypoxic perfusate during the

preconditioning protocol abrogates IPC’s protection (Dost

et al., 2008) The identity of the ROS species involved has not

been positively identified but seems to be a downstream

product of HO· and is likely a product of phospholipid

oxi-dation (Garlid et al., 2013).

Adenosine signalling

Signalling initiated by the third endogenous agonist that

triggers IPC, adenosine, is different Adenosine’s

cardiopro-tective effect is not dependent on Src tyrosine kinase or PI3K

(Qin et al., 2003) Adenosine signalling seems to completely

bypass mtKATPand ROS production (Cohen et al., 2001) and it

more directly activates PKC (see Figure 1) which is where all

of the trigger signalling converges The adenosine A1receptor

is coupled through Gito PLC and PLD After the ligand binds

to the receptor, Gi is cleaved intoα and βγ moieties which

activate PLC in the sarcolemma This enzyme catalyzes the

hydrolysis of membrane inositol-containing phospholipids,

including phosphatidylinositol 4,5-bisphosphate The

result-ing DAG stimulates translocation and activation of PKC PLD

also increases DAG levels by degrading phosphatidylcholine

into choline and phosphatidic acid and the latter is

trans-formed by a phosphohydrolase into DAG These

phospho-lipid activators of PKC also trigger release of zinc from PKC’s

regulatory domain (Korichneva et al., 2002) The diversity of

signalling among the triggers is confusing, but also

reassur-ing The redundancy ensures cardioprotection even if one or

more elements in the triggering cascade are blocked

The mediator phase

All signalling to this point occurs during the preconditioning

cycles of ischaemia and reflow These steps are collectively

called the trigger phase Subsequent steps, and there are

several, are part of the mediator phase which occurs

follow-ing termination of the prolonged period of ischaemia (the

index ischaemia) with reperfusion (Figure 1)

A2B receptors

It had been noted that adenosine receptors were required forIPC’s protection in the mediator phase One hypothesis sug-gested that preconditioning is protective by increasing tissueadenosine levels through activation of ecto-5′-nucleotidase

(Kitakaze et al., 1993) However, measurements of myocardial

adenosine levels revealed that tissue adenosine concentration

actually falls in IPC hearts (Goto et al., 1996; Martin et al.,

1997) Our studies have indicated that the initial step of themediator phase is activation of adenosine A2B receptors

(Philipp et al., 2006) This receptor has a very low affinity for

adenosine such that even during ischaemia when tissueadenosine levels reach 1–4μM, this level would still be wellbelow the A2Badenosine receptor’s KDof 5–15μM However,PKC activation appears to raise the affinity of the A2Breceptorpermitting the adenosine concentration in ischaemic myo-cardium to be sufficient for occupation of this receptor (Kuno

et al., 2007) It had already been shown that PKC activity can

sensitize A2Bsignalling, although no physiologic significance

was attributed to the observation (Nordstedt et al., 1989; Nash et al., 1997; Trincavelli et al., 2004) Although the

details of this sensitization are still unknown, it would appear

A2Breceptors can respond to the heart’s endogenous sine only after this sensitization Thus, we proposed that theaffinity state of the A2Breceptor is the determinant that dis-tinguishes the preconditioned from the non-preconditionedphenotype Our observation of involvement of the A2Brecep-

adeno-tor in IPC was supported by Eckle et al (2007) who studied

mice genetically modified to lack one of the four adenosinereceptor subtypes While A1, A2A and A3adenosine receptorknockout mice could be preconditioned, A2Bknockout micecould not However, there is evidence suggesting a coopera-tive role of A2Aand A2Badenosine receptors in some forms of

cardioprotection (Xi et al., 2009; Methner et al., 2010).

The reperfusion injury survival kinases (RISK) pathway

A kinase cascade involving PI3K, Akt and ERK has been posed to occur in the first minutes of reperfusion followingthe index ischaemia (Hausenloy and Yellon, 2004; Hausenloy

pro-et al., 2005) These kinases have collectively been termed RISK

(Hausenloy and Yellon, 2004) Although RISK are clearly

involved in cardioprotection in rat (Hausenloy et al., 2005) and rabbit (Yang et al., 2004b; 2005) hearts, their involve-

ment may not be universal In pig hearts, RISK are less

impor-tant (Skyschally et al., 2009a) A distinct alternate pathway

utilizing membrane TNF-α receptors and cytoplasmic JAKand STAT has been proposed (see succeeding text), althoughthe end-effector for this and the RISK pathways appears to beidentical

IPC’s end-effector

IPC’s end-effector appears to be the mitochondrial ity transition pore (mPTP) and its inhibition is considered to

permeabil-be the final step in the protective signal transduction

pathway (Griffiths and Halestrap, 1993; 1995; Squadrito et al., 1999; Di Lisa et al., 2001; Hausenloy et al., 2002) Although

the molecular structure of mPTP is controversial, when

formed it is a high conductance pore in the inner

mitochon-drial membrane that dissipates the transmembrane proton/

electrochemical gradient that drives ATP generation The

presence of the pore would logically lead to ATP depletion,

enhanced ROS production, failure of membrane ion pumps,

solute entry, organelle swelling and ultimate mitochondrial

rupture Destruction of large numbers of mitochondria will

result in necrosis of the cardiomyocyte Importantly, mPTP

formation is inhibited by acidosis and promoted by calcium

and ROS The low pH during ischaemia inhibits transition

pore formation But restoration of pH coupled with rapid

elevation in mitochondrial calcium and ROS cause the pores

to form soon after reperfusion

The cardioprotective signalling pathways keep mPTP

closed In the RISK pathway, there is involvement of an

addi-tional intervening kinase, glycogen synthase kinase-3β

(GSK-3β) (Tong et al., 2002; Gross et al., 2004; Juhaszova et al.,

2004) This kinase is likely the final cytoplasmic kinase in

IPC’s signal transduction pathway Interestingly,

precondi-tioning leads to Ser9 phosphorylation and inhibition, not

activation, of this kinase Thus, GSK-3β inhibition blocks

mPTP formation Accordingly, GSK-3β inhibitors given at

rep-erfusion mimic preconditioning (Förster et al., 2006).

Alternative signalling pathways

As already noted, the importance of RISK has been clearly

demonstrated in rat (Hausenloy et al., 2005) and rabbit (Yang

et al., 2004b; 2005) hearts, but their involvement may not be

required in all species In a well-established pig heart model,

activation of RISK was not increased by ischaemic

postcondi-tioning (IPoC), protection mediated by several brief

reocclu-sions after release of the prolonged index coronary occlusion,

over that seen in control hearts without postconditioning

(Skyschally et al., 2009a) Furthermore, wortmannin, a potent

antagonist of PI3K, could not abort postconditioning’s

pro-tection (Skyschally et al., 2009a) In response to this

conun-drum, additional investigations uncovered another signalling

pathway not dependent on RISK

Survivor activating factor enhancement

(SAFE) pathway

The SAFE pathway has been established, at least in rodent

(Lecour et al., 2005b; Lacerda et al., 2009; Lecour, 2009) and

porcine (Bhatt et al., 2013) hearts However, a caveat is

impor-tant Some individual signalling steps have been identified,

but a roadmap or signal transduction pathway as developed

for the RISK pathway (Figure 1) is not yet available So

evi-dence supporting involvement of the SAFE pathway is more

fragmentary, even if compelling

TNF-α signalling

The cytokine TNF-α is an important endogenous

cardiopro-tectant released by IPC (Smith et al., 2002; Lecour, 2009) and

IPoC (Lacerda et al., 2009), possibly as part of the myocardial

inflammatory response during reperfusion TNF-α knockout

mice cannot be protected by either IPC (Smith et al., 2002;

Lecour, 2009) or IPoC (Lacerda et al., 2009; Lecour, 2009),

whereas low-dose exogenous TNF-α in lieu of ischaemia can

both precondition (Smith et al., 2002; Lecour et al., 2005b; Lecour, 2009) and postcondition (Lacerda et al., 2009; Lecour,

2009) hearts As seen with the Gi-coupled receptor triggeringdescribed earlier for IPC, TNF-α preconditioning of rat heartscan be abolished by the antioxidant N-2-mercaptopropionylglycine (Lecour et al., 2005a), a ROS scavenger, 5-

hydroxydecanoate (Lecour et al., 2002), an antagonist of

mtKATP, and chelerythrine (Lecour, 2009), a PKC antagonist,implying free radicals, mtKATPchannels, and PKC play impor-tant roles However, further information about the down-stream effect of ROS, opening of mtKATP, or PKC or theirtargets is not available Interestingly, TNF-α’s effect on ischae-mic myocardium is concentration-dependent High doses ofTNF-α are not protective and may actually increase infarct

size (Lecour et al., 2002; Lecour, 2009).

There are two TNF receptor isoforms, TNFR1 and TNFR2.Exogenous TNF-α confers cardioprotection in TNFR1 (alsoknown as TNFRSF1A) but not TNFR2 (also known asTNFRSF1B) knockout mice, thereby implying it is TNFR2which is responsible for the ligand’s cardioprotective effect

(Lacerda et al., 2009) TNF-α administered as either a pre- or

postconditioning-mimetic does not lead to phosphorylation

of either Akt or ERK, and neither PD98059 nor wortmannin,antagonists of the ERK and PI3K pathways, respectively, canabort the protection of exogenous TNF-α The SAFE path-way’s downstream signalling is, therefore, not dependent on

these traditional RISK (Lecour et al., 2005b; Lacerda et al.,

2009) In contrast, TNF-α, IPC and IPoC all phosphorylateSTAT3 and the protective effect of pharmacological pre- andpostconditioning with TNF-α is abolished by AG490, an

inhibitor of STAT3 (Lecour et al., 2005b; Lacerda et al., 2009).

JAKs are a family of tyrosine kinases associated with thecytoplasmic domains of cytokine and growth factor recep-tors, for example, IL-6, growth hormone and TNFR2 Afterthe TNF-α ligand binds to its receptor, two adjacent JAKs aretransphosphorylated and subsequently activate STAT pro-teins by phosphorylation Tyrosine-phosphorylated STATproteins form homo- and heterodimers that translocate tothe nucleus where they influence gene transcription, espe-cially of stress-responsive genes (Levy and Lee, 2002; Myers,2009) Serine-phosphorylated STAT translocates to mitochon-dria to regulate electron transport (Myers, 2009; Wegrzyn

et al., 2009) Although STAT3 is by definition a transcription

factor, its effects in ischaemia/reperfusion are much too rapid

to assume that it is working by modulating gene tion Therefore, it must have additional direct effects Itappears to protect by phosphorylating and, therefore, inacti-vating GSK-3β (Lacerda et al., 2009; Pedretti and Raddatz,2011), also a downstream target in the RISK pathway Thus,the RISK and SAFE pathways appear to converge on the sametargets In fact there is some evidence of cross-talk betweenthese two pathways, so they may not be totally independent

transcrip-(Lecour, 2009; Somers et al., 2012) Additionally, IPoC in pigs

increased tyrosine phosphorylation of mitochondrial STAT3which improves complex I respiration and calcium retention

capacity (Heusch et al., 2011) Inhibition of JAK/STAT blocks

both increased phosphorylation of mitochondrial STAT3 andthe cardioprotective effect of IPoC Mitochondrial STAT3co-immunoprecipitates with cyclophilin D, the target of themPTP inhibitor cyclosporin A and, therefore, could inhibit

pore formation (Boengler et al., 2010).

BJP

Pre- and postconditioning signalling

Sphingosine is a trigger for RISK and SAFE

Sphingosine is a membrane sphingolipid which is catalyzed

to sphingosine 1-phosphate (S1P) by two sphingosine kinase

(SPHK) isoforms, SPHK1 and SPHK2 It is the former that is

associated with cell survival S1P is released in both IPC and

IPoC (Karliner, 2013) Many S1P actions are mediated

through S1P GPCR subtypes The S1P1receptor is most

promi-nently expressed in cardiomyocytes The S1P1 receptor

couples to Gαi S1P2and S1P3 receptors are also present on

cardiomyocytes and couple to both Gαqand Gαi Binding of

the ligand S1P to the S1P1 receptor leads to downstream

activation of ERK1/2 and S1P3 receptor binding results in

activation of PI3K and Akt Thus, S1P cardioprotection in part

depends on RISK (Knapp, 2011; Somers et al., 2012).

However, S1P cardioprotection is also dependent on the SAFE

pathway through the S1P2receptor which activates ERK1/2

and subsequently STAT3 (Knapp, 2011; Somers et al., 2012).

As already noted, multiple pathways provide potential for

robust protection

Sphingosine intermediates are also involved in

cardiopro-tection mediated by TNF-α The latter’s protective effect is

attenuated in the presence of inhibitors of the sphingolipid

pathway (Lecour et al., 2002) TNF receptor-associated factor

2 (TRAF2) is a downstream target of TNFR2 TRAF2 can

acti-vate intracellular formation of S1P by up-regulating SPHK1

(Frias et al., 2012) Also, S1P activates STAT3.

This redundancy of pathways probably enhances the

potential survival of the cell Blockade of any one pathway

does not lead to inevitable death of the cell It is still possible

for an alternate pathway to provide some protection

Alter-natively, we may simply be looking at isolated sections of a

larger complex integrated system that we still do not fully

appreciate This may be analogous to the fable of the blind

men describing an elephant based only on the part of the

animal they were touching

Genesis of reperfusion therapy

Hence, IPC is a potent cardioprotective intervention that is

the result of a complex, two-phase signalling pathway

leading to inhibition of mPTP formation However, the

obvious drawback is that IPC by definition must be instituted

prior to the onset of ischaemia In patients presenting to the

hospital with an AMI, ischaemia is already ongoing and

pre-conditioning is not possible However, Hausenloy et al.

(2005) proposed that if IPC, an intervention introduced

before the onset of ischaemia, protects by inducing activation

of the RISK pathway at reperfusion, then it should still be

possible to activate this pathway during ischaemia and still

effect salvage of myocardium This revolutionary paradigm

shift provided hope that IPC could be translated into a

mean-ingful clinical intervention by focusing on early reperfusion

Indeed, multiple reagents were found to protect the

myocar-dium when given in the first minutes of reperfusion, for

example, insulin (Baines et al., 1999), the adenosine A1/A2

agonist AMP579 (Xu et al., 2000), the A2Badenosine

receptor-selective agonist BAY 60-6583 (Albrecht et al., 2006), TGFβ1

(Baxter et al., 2001), urocortin (Schulman et al., 2002),

cardiotrophin-1 (Liao et al., 2002), adenosine agonist

5′-(N-ethylcarboxamido) adenosine (Yang et al., 2004a), bradykinin (Yang et al., 2004a), natriuretic peptides (Baxter, 2004; Yang

et al., 2006a), erythropoietin (Cai and Semenza, 2004; Parsa

et al., 2004) and cyclosporin A (Hausenloy et al., 2009) All

depend on activation of PI3K and/or ERK except for sporin A which is a direct inhibitor of mPTP formation

cyclo-Clinical trials

Not surprisingly, several clinical trials of proposed mimetics have been completed, although none has beengreatly successful These trials require study of many patientsand are expensive Repeated failures have left the pharmaceu-tical companies quite leery Two large clinical trials, Amistad

IPC-I (Mahaffey et al., 1999) and IPC-IIPC-I (Ross et al., 2005), were

organ-ized to evaluate the effectiveness of adenosine In the firsttrial, all patients with AMI were evaluated, whereas in thesecond trial, patients with only higher risk anterior infarctswere included In Amistad I, there was no difference between

the control and adenosine-treated subjects, although a post

hoc analysis suggested that data in the subgroup with anterior

infarcts looked promising (Birnbaum et al., 2002) In Amistad

II, results were again disappointing Smaller infarcts werenoted in only a high-dose subgroup, but clinical outcomeswere not improved Although adenosine plays an importantrole in preconditioning as both a trigger and a mediator, theAmistad trials used low-dose i.v infusion of adenosine which

in preclinical studies had clearly not been universally

success-ful at protecting ischaemic myocardium (Olafsson et al., 1987; Goto et al., 1991; Norton et al., 1991; 1992; Pitarys

et al., 1991; Velasco et al., 1991; Vander Heide and Reimer,

1996; Budde et al., 2000) It was probably not advisable to

undertake such large and expensive trials until the cause ofthe discrepant data had been identified and resolved Adeno-sine’s hypotensive side effect limits the concentration thatcan be administered parentally and we were unable to pre-condition rabbit hearts with the highest dose of i.v adeno-

sine they would tolerate (Liu et al., 1991) We could, however,

condition them with receptor-selective adenosine analogs

such as AMP579 (Xu et al., 2003).

Atrial natriuretic peptide activates PKG in cardiomyocytesand mimics IPC when injected just prior to reperfusion in

animals (Yang et al., 2006a) It produced a statistically

signifi-cant, but very modest, reduction in infarct size and a similarly

modest increase in ejection fraction (Kitakaze et al., 2007).

The disappointingly modest effect might be explained byfailure to stratify patients into low- and high-risk groups Thesize of a patient’s ischaemic zone is dependent on the loca-tion in the coronary artery where the thrombus forms

Studies from Ovize’s laboratory (Staat et al., 2005) indicate

that in patients with AMI who are reperfused with primaryangioplasty, those with small ischaemic zones have verysmall infarcts and virtually complete recovery regardless oftreatment Including these patients in the analysis greatlydilutes the potential significance of any intervention Thera-peutic benefit can best be appreciated in the subgroup ofhigh-risk patients presenting with large ischaemic zones.Other possible reasons for the modest result are discussed inthe succeeding text

During ischaemia, pH falls as H+accumulates As a result,the Na+/H+exchanger (NHE) is activated and Na+exchangesfor H+in a 1:1 stoichiometric manner In turn, the Na+/Ca2+

exchanger will transport Na+out of the cell in favour of Ca2+.

These ionic movements are magnified during reperfusion

when restored blood flow quickly normalizes the pH of the

extracellular fluid The resulting high intracellular Ca2 +

con-centration should encourage mPTP formation It was decided

to evaluate NHE blockers cariporide (Théroux et al., 2000;

Mentzer et al., 2008) and eniporide (Zeymer et al., 2001) in

large clinical trials despite preclinical investigations which

demonstrated efficacy only when the drug was administered

before ischaemia (Miura et al., 1997) In the first trial of

cari-poride, GUARDIAN, patients studied had unstable angina

pectoris, non-ST-segment elevation myocardial infarction,

angioplasty or coronary revascularization surgery (Théroux

et al., 2000) Only those that had pretreatment, the surgical

group, showed any benefit A second trial, EXPEDITION,

con-centrated on coronary bypass patients, but the new study

design inexplicably included prolonged infusions of

cari-poride which were associated with more strokes (Mentzer

et al., 2008) The increased stroke risk doomed further

con-sideration of cariporide Although eniporide, another NHE

blocker, was evaluated in patients with acute

ST-segment-elevation myocardial infarction (STEMI), no difference was

observed (Zeymer et al., 2001).

Investigations of other interventions which may have

shown some promise in preclinical evaluations have also

not been very successful Thus, pexelizumab (APEX AMI

Investigators et al., 2007), an antibody to a complement

com-ponent, erythropoietin (Cleland et al., 2010), a stimulator of

haematopoiesis in response to hypoxia, and delcasertib

(Lincoff et al., 2014), a selective inhibitor of PKCδ, all failed to

meet the primary objectives of the trials, reduction of infarct

size and improvement of the clinical status of the subjects

There is a lesson: clinical trials should probably not be

under-taken until multiple preclinical laboratories have confirmed

salutary effects of the intervention and until practical

infor-mation about dosing and timing of administration has been

established (Downey and Cohen, 2009)

A small proof-of-concept study of cyclosporin A in

patients with AMI produced very encouraging results (Piot

et al., 2008) Patients were stratified by the size of their

ischae-mic zone and each received a bolus of cyclosporin A before

recanalization Those with the highest risk benefited the most

from exposure to the drug There are plans to repeat this

investigation in a much larger cohort in Europe However, as

explained in the succeeding text, a second study may be

problematic because concomitant use of antiplatelet drugs

dictated by current standard of care considerations may mask

cyclosporin’s protection

There have been other proof-of-concept studies

examin-ing commonly used agents Van de Werf et al (1993) studied

the effect of atenolol administered prior to thrombolysis in

patients with AMI Thisβ-blocker had no impact on infarct

size, a result echoing that of a study in dogs by Reimer and

Jennings (1984) Nonetheless, a very recent examination of

i.v metoprolol shortly before percutaneous coronary

inter-vention (PCI) in patients with STEMI was encouraging

(Ibanez et al., 2013) This β-blocker modestly decreased

infarcted myocardium as a percentage of risk zone from

approximately 78% in untreated hearts to 68% Although

there was protection, small group sizes limit the significance

of the conclusion and require conduct of a large, expensive

clinical trial for confirmation There is no evidence thatβ-adrenoceptor blockade triggers IPC signalling as detailedearlier The same is true for exenatide, a glucagon-like

peptide-1 (Lønborg et al., 2012) IPC’s signalling is not the

only way to protect the heart against infarction, and thermia and early reperfusion are obvious examples

hypo-IPoC

It was clearly understood that preconditioning cycles must be

completed before initiation of ischaemia Yet Hausenloy et al (Hausenloy and Yellon, 2004; Hausenloy et al., 2005) found

that IPC actually exerted its protection at reperfusion This

finding led Vinten-Johansen et al to test whether serial

coro-nary occlusions after the index coronary occlusion/reperfusion might also protect the ischaemic heart Aftermany tries, they found that several (three) short (30 s) cycles

of reperfusion/occlusion immediately after the initial fusion were almost as protective as IPC in an open-chest dog

reper-model (Zhao et al., 2003) They called this IPoC This

seem-ingly improbable observation has been reproduced in many

laboratories (Skyschally et al., 2009b) and the ensuing

protec-tion was shown to be dependent on the same signals as IPC

(Yang et al., 2004b; 2005).

Again, the final effector for IPoC appeared to be

preven-tion of mPTP formapreven-tion (Argaud et al., 2005; Gateau-Roesch

et al., 2006) Because of the widespread success in the

experi-mental laboratory, a leap was made to the clinical arena.Patients with acute STEMI have thrombotic occlusion of acoronary artery Standard treatment is recanalization, usually

by mechanical aspiration or pulverization of the thrombus bypercutaneous transluminal coronary angioplasty (PTCA).Opening of the occluded coronary artery by PTCA is equiva-lent to removing the ligature around the snared coronaryartery in the experimental animal For patients treated withprimary angioplasty, IPoC could be accomplished by repeatedballoon inflations to interrupt reflow for the postcondition-ing cycles The initial report of IPoC in the cardiac catheteri-

zation laboratory was very encouraging (Staat et al., 2005).

Using a risk stratification design, they showed a highly nificant reduction of infarct size in IPoC patients with largeischaemic zones But why does staccato reperfusion lead tomyocardial salvage?

sig-In the non-conditioned nạve heart following coronaryocclusion, mtKATP open during ischaemia, but there is nooxygen so the pathway is blocked at the redox signalling step.During ischaemia, mPTPs are inhibited by acidosis in thetissue probably by blocking calcium binding to cyclophilinand displacing the latter which is required for mPTP forma-tion Upon reperfusion, acids quickly wash away restoring pH

to 7.4 and mPTP forms before PKC can be activated to triggerthe remainder of the signalling pathway thus resulting innecrosis of the tissue On the other hand, IPoC maintainssome acidosis in the reperfused tissue because of the repeatedocclusion periods while still allowing oxygenation during the

reperfusion periods (Cohen et al., 2007b; 2008)

Reintroduc-tion of oxygen while the tissue pH is still acidic allows thetissue to activate PKC through redox signalling while mPTP

formation is still inhibited (Cohen et al., 2007b; 2008) Once

the PKC pathway is activated, the cell is able to inhibit mPTP

BJP

Pre- and postconditioning signalling

formation through the conditioning pathway that IPC uses

even after pH is normalized, and hence necrosis is reduced

Thus, there is a race between ROS-mediated activation of

PKC leading to subsequent triggering of the remainder of the

signal cascade and washout of mPTP-inhibiting H+ Figure 2

summarizes the pH hypothesis of IPoC’s protection mPTPs

in the nạve, non-conditioned heart (Figure 2, upper panel)

are inhibited by the low pH during the ischaemic period But

as soon as reperfusion is permitted, H+ is washed out and

mPTPs open leading to tissue necrosis In IPC (Figure 2,

middle panel), signalling up to the opening of mtKATPoccurs

during the first brief ischaemic period During the brief

rep-erfusion oxygen is resupplied which leads to ROS generation

and activation of PKC which then can sensitize adenosine

receptors Thus, at the beginning of reperfusion following the

prolonged index coronary occlusion, RISK are activated and

mPTPs are inhibited The bottom panel of Figure 2 depicts

events in IPoC The initial signalling up to opening of mtKATP

occurs during the prolonged period of ischaemia, but

signal-ling cannot proceed until reperfusion when oxygen is

rein-troduced into the ischaemic tissue leading to generation of

ROS Because of the limited reflow, pH only partially recovers

The low pH inhibits mPTP formation long enough until

redox signalling can lead to adenosine receptor population

with sensitization and subsequent RISK signalling that results

in inhibition of mPTP formation even after the muscle is fullyreperfused

The pH hypothesis is consistent with other observationsmade in experimental animals Reperfusion interventionsmust be applied in the first minutes of reperfusion Thus,

delayed IPoC (Yang et al., 2004b; Philipp et al., 2005) or late infusion of the cardioprotective AMP579 (Xu et al., 2003)

leads to loss of the cardioprotective effects Presumably, suchdelay would permit pH normalization before initiation of theintervention, thus allowing mPTP formation Once thisoccurs, no intervention dependent for its success on keepingmPTP closed would be expected to salvage myocardium inthe risk zone Also, simply reperfusing the heart for several

minutes with low pH buffer mimics IPoC (Cohen et al.,

Adenosine

mPTP (Low pH effect)

Receptor PI3-K eNOS PKG mtKATP

pH

PI3-K ERK

Ischemia

Figure 2

Signalling during ischaemic pre- and postconditioning and effect of pH and transient reoxygenation on that signalling and mPTP formation See

Figure 1 for abbreviations Modified from Cohen et al (2007b; 2008).

principally sevoflurane, isoflurane and desflurane were noted

to also have cardioprotective abilities when applied in lieu of

the brief cycles of ischaemia/reperfusion either before the

prolonged index coronary occlusion (preconditioning) (Cope

et al., 1997) or following it (postconditioning) (Chiari et al.,

2005) The signalling steps are not as clearly defined as in IPC

and IPoC, but it is fair to say there are many parallels

Basi-cally, the volatile gases signal through adenosine and opioid

receptors, modulate G proteins, stimulate PKC and other

intracellular kinases, open mtKATP channels leading to ROS

generation and activate RISK to keep mPTP from forming

(Tanaka et al., 2004; Chiari et al., 2005; Feng et al., 2005;

Pravdic et al., 2009) The gases may also have more direct

effects on mtKATP The potential clinical impact is obvious,

although utility is limited to the surgical suite, either cardiac

or non-cardiac (Swyers et al., 2014).

Platelets and cardioprotection

As noted earlier, one of the earliest cardioprotective

interven-tions applied clinically was IPoC The initial clinical report

was very encouraging (Staat et al., 2005) and the intervention

was adopted by many cardiac catheterization laboratories,

partly because of the ease of application and partly because of

the anticipated small likelihood of complications Despite

this early enthusiasm, other clinicians who attempted to

rep-licate the positive results could not (Sörensson et al., 2010;

Freixa et al., 2012; Tarantini et al., 2012; Hahn et al., 2013;

Limalanathan et al., 2014) Would this intervention have to

join all of the other interventions which initially showed

great promise but which did not live up to expectations and

which failed to produce a significant and consistent

benefi-cial clinical effect? Could there be a logical explanation for

the inability of these later studies of IPoC to reproduce the

early encouraging data?

Platelets are important cellular elements for initiation and

propagation of a thrombus It is now universally accepted

that STEMI is caused by thrombosis and coronary occlusion

following rupture of a plaque Exposure of circulating

plate-lets to collagen leads to their activation and accumulation

Platelet-activating factor (PAF) is a phospholipid which is

released by neutrophils and monocytes during oxidative

stress or ischaemia/reperfusion (Penna et al., 2011) PAF is a

chemoattractant for platelets and neutrophils and

predis-poses to capillary plugging and release of proteolytic enzymes

and inflammatory mediators Additionally, it causes coronary

vasoconstriction Cardiomyocytes also produce PAF and the

latter’s synthesis is triggered by ROS generation and oxidative

stress at the beginning of reperfusion PAF binds to receptors

located on various cell types including smooth muscle cells,

cardiomyocytes and endothelial cells PAF receptor

antago-nists limit infarction in models of ischaemia/reperfusion

(Montrucchio et al., 1990; Ma et al., 1992) Curiously, low

doses of PAF are paradoxically cardioprotective (Penna et al.,

2005; 2011) The PAF receptor is a Giprotein-coupled receptor

that triggers signalling similar to that seen in IPC (Figure 1)

Because the major risk of intracoronary dilatation and

stenting is stent thrombosis and occlusion, antiplatelet drugs

were tested as anticoagulants in patients undergoing primary

angioplasty for AMI Many clinical studies have

demon-strated that antiplatelet agents do indeed improve prognosis

of patients after AMI and minimize complications of stenting

(Antiplatelet Trialists’ Collaboration, 1994; Yusuf et al., 2001; Sabatine et al., 2005a,b; Wiviott et al., 2007; Wallentin et al., 2009; Bhatt et al., 2013) This protection is particularly

evident when the antiplatelet drug is given as a loading doseprior to the recanalization procedure Thus, the COX antago-nist aspirin, the thienopyridines clopidogrel and prasugreland the triazolopyrimidines ticagrelor and cangrelor are veryeffective agents and all except cangrelor have won regulatoryapproval and have become standard of care in the treatment

of patients with AMI or stenting Aspirin blocks production ofthromboxane by the platelet, while the thienopyridines andtriazolopyrimidines block the platelet P2Y12ADP receptor andall effectively attenuate platelet aggregation It has beenassumed that it is the anti-aggregatory effect of these agents

on platelets to minimize intravascular thrombosis that isresponsible for their well-documented clinical benefits.However, that mechanism of action has not been unequivo-cally determined In this regard, it is instructive to reviewsome of the preclinical data

Preclinical studies of antiplatelet drugs

Barrabés et al (2010) noted that activated platelets from

patients with AMI infused into isolated rat hearts before onset

of ischaemia/reperfusion increased infarct size, whereas lets from healthy volunteers had no effect Furthermore, per-fusion of previously ischaemic hearts with platelets activated

plate-in a second animal followplate-ing ischaemia/reperfusion led todeterioration of left ventricular function and larger myocar-

dial infarcts (Knight et al., 2001; Mirabet et al., 2002)

Aggre-gation of platelets from mice with deficiencies of either

platelet receptor glycoprotein (GP) VI (Takaya et al., 2005; Li

et al., 2007) or signalling protein Gq (Weig et al., 2008) is

diminished, and infarcts are smaller following ischaemia/reperfusion These observations suggest activated plateletshave deleterious effects on ischaemic myocardium andprovide some support for the hypothesis that there is a rela-tionship between platelet aggregation and consequences ofmyocardial ischaemia/reperfusion, for example, infarct size.Preclinical investigations of blockade of platelet aggrega-tion in ischaemia/reperfusion have mostly studied effects ofGPIIb/IIIa antagonists An experimental GPIIb/IIIa inhibitor

which abolished in vitro platelet aggregation decreased

infarc-tion in dogs undergoing ischaemia/reperfusion when it was

administered before reperfusion (Kingma et al., 2000) However, Kingma et al (2000) also noted that the platelet

antagonist had no effect on myocardial blood flow duringreperfusion, and therefore, postulated that this infarct-sparing action was not related to blood flow but rather wasthe result of a direct protective effect on heart muscle Thiswas the first suggestion of a direct cardioprotective effect by

an inhibitor of platelet aggregation

Kunichika et al (2004) made similar observations in dogs

treated with tirofiban, a GPIIb/IIIa antagonist However, thisagent which decreased infarct size also increased myocardialblood flow within the risk area Consequently, the investiga-tors attributed the agent’s cardioprotection to improvement

in microvascular flow Tirofiban also decreased the area ofno-reflow in pigs during reperfusion and decreased infarct

size (Yang et al., 2006b) In dogs with coronary thrombosis

BJP

Pre- and postconditioning signalling

treated with angioplasty, tirofiban improved myocardial

blood flow following reperfusion, decreased the size of the

no-reflow zone and made infarcts smaller (Sakuma et al.,

2005) It was assumed that inhibition of platelet aggregation

protected by preventing microthromboembolism

Additional studies failed to corroborate this hypothesis

The deleterious effect of the addition of activated pig platelets

to perfused, isolated rat hearts subjected to ischaemia/

reperfusion was not blocked by tirofiban (Mirabet et al.,

2002) A second GPIIb/IIIa inhibitor had no effect on infarct

size in a porcine model of ischaemia/reperfusion (Barrabés

et al., 2002) In both studies, platelet aggregation was blocked

by the GPIIb/IIIa antagonists It is not known why these latter

studies differed from the former

P2Y12 receptor inhibitors may be

postconditioning agents

Because of this confusion, we evaluated a variety of platelet

inhibitors in rabbits undergoing 30 min coronary occlusion/

3 h reperfusion (Yang et al., 2013c) For most of the studies,

we examined the effects of cangrelor, an i.v agent which

could be administered minutes before reperfusion and which

would have immediate effects Oral agents suffer from the

limitations imposed by intestinal absorption and the

require-ment for conversion of the administered pro-drug clopidogrel

or prasugrel to active metabolites This delay causes an

uncer-tainty of timing of onset of biological effect A cangrelor

bolus of 60μg·kg−1followed by an infusion of 6μg·kg−1·min−1

attenuated platelet aggregation by more than 94% Cangrelor

resulted in an impressive decrease in infarct size from 38% of

the risk zone in control rabbits to 19%, similar to the degree

of protection seen after IPoC Delay in cangrelor

administra-tion until 10 min after release of the coronary occlusion led

to abrogation of protection, similar to that seen with delayed

IPoC (Yang et al., 2004b; Philipp et al., 2005).

As described earlier, IPoC’s protection is known to depend

on a complex signal transduction pathway Accordingly, we

tested seven inhibitors of IPoC’s signalling and protection:

wortmannin and LY294002 (PI3K/Akt antagonists), PD98059

(antagonist of MAPK kinase 1/2 and therefore, ERK 1/2),

5-hydroxydecanoic acid (putative blocker of mtKATP),

8-(p-sulfophenyl) theophylline (non-selective antagonist of

adenosine receptors), MRS 1754 (selective antagonist of

adenosine A2Breceptors) and N-2-mercaptopropionylglycine

(scavenger of ROS and blocker of redox signalling) All

abol-ished cangrelor’s protection However, importantly, none

restored platelet reactivity Therefore, cangrelor’s

anti-aggregatory effect was still intact, but its cardioprotective

action was totally blocked

Hence, IPoC and cangrelor have identical kinase and

receptor ‘fingerprints’ and this conclusion strongly supports

the contention that the signalling and mechanism of

protec-tion of the two intervenprotec-tions are the same We also

deter-mined whether the combination of cangrelor and IPoC

would have an additive protective effect It did not, further

supporting the assumption that both induce protection by

the same mechanism (Yang et al., 2013c) Of course, to make

this conclusion, it is critical that the effect of each individual

intervention is maximized Obviously, there would be an

additive effect of two agents using the same pathway if one or

both was used at a submaximal concentration Hence, we

propose that cangrelor is a bona fide conditioning agent andsignal transduction rather than any effect on thrombosiscauses the protection Yet, we found that the magnitude ofprotection was correlated with the degree of suppression ofaggregation indicating that P2Y12blockade was common toboth processes

Cangrelor in both primate (Yang et al., 2013b) and rodent (Yang et al., 2013a) hearts was also very protective when

administered just before reperfusion Cangrelor’s protection

was equivalent to that of IPoC in macaque hearts (Yang et al., 2013b) and IPC in rat hearts (Yang et al., 2013a) In monkeys,

an antibody to platelet GPVI also decreased infarct size, cating another intervention which decreased platelet aggre-

indi-gation and infarction (Yang et al., 2013b).

Clopidogrel, a widely used P2Y12antagonist in patientswith AMI and PTCA, was fed to rabbits for 2 days and it

blocked platelet aggregation by 78% (Yang et al., 2013c).

Clopidogrel-treated animals also had significantly smallerinfarcts than untreated rabbits Wortmannin and MRS 1754each abolished the protective effect, but the drug’s anti-aggregatory action remained intact

Finally, ticagrelor, a third platelet P2Y12receptor nist, administered by gavage to rats 2 h before coronaryocclusion, decreased infarction from 45% of the risk zone in

antago-control animals to 26% (Yang et al., 2013a) This protective

effect was predictably blocked by wortmannin and PKCantagonist chelerythrine Neither blocker interfered withticagrelor’s inhibitory effect on platelet reactivity

Therefore, several pharmacologic and biologic tions that blocked platelet aggregation also spared ischaemicmyocardium from infarcting It is critical to realize thatalthough an antiplatelet effect linked all of the agents, inhi-bition of platelet activity was not the determining factor forcardioprotection These appear to be true conditioningagents Cangrelor in isolated rabbit hearts perfused withplatelet-free Krebs buffer was not protective, suggesting thatsome blood element, presumably platelets, is somehowinvolved Indeed, cangrelor’s protective effect was lost in ratsmade thrombocytopenic with anti-thrombocyte serum(unpublished observation)

interven-Are today’s patients already postconditioned

by loading doses of antiplatelet drugs?

In addition to the possible confounding effects ofco-morbidities in man, generally absent in animal models,these experimental data on antiplatelet interventions, specifi-cally P2Y12 receptor antagonists, may help to explain theobservations of protection or lack of protection followingclinical IPoC Because P2Y12 antagonists are conditioningagents, they themselves would already be protecting theheart Therefore, addition of a second intervention as IPoCwhich protected by the same mechanism would be expected

to have little additional effect, similar to our observations inrabbits in which cangrelor and IPoC together were no more

protective than either alone (Yang et al., 2013c) In the later

clinical studies of IPoC, virtually, all patients had received

clopidogrel before the intervention (Sörensson et al., 2010; Freixa et al., 2012; Tarantini et al., 2012; Hahn et al., 2013; Limalanathan et al., 2014), thus marking the patients as pro-

tected and perhaps dooming IPoC to be a superfluous andunneeded intervention However, in the initial study by

Ovize’s group in which patients with AMI were recruited

prior to 2005, only about half had been premedicated with

clopidogrel (Staat et al., 2005) At our urging, these

investiga-tors went back to their database and segregated patients

according to pre-IPoC administration of clopidogrel (Roubille

et al., 2012) Control patients treated with clopidogrel had

smaller infarcts than those who were untreated, supporting

our conclusion that clopidogrel is a cardioprotective agent

But these authors also noted that the combination of

clopi-dogrel and IPoC salvaged more tissue than IPoC, contrary to

our observations in rabbits However, it is likely that neither

intervention, clopidogrel nor IPoC, was optimized to fully

condition the hearts when applied individually in contrast to

our ability to maximize the effect of each intervention in

animal models Most of the patients in Roubille’s

retrospec-tive analysis (Roubille et al., 2012) had received only 300 mg

of clopidogrel which is clearly suboptimal (Patti et al., 2011).

Also, the optimal postconditioning protocol for human

hearts is unknown and only one protocol was tested If

neither intervention was optimal, an additive effect would be

expected

Piot et al (2008) reported a significant reduction in infarct

size by cyclosporin A in patients undergoing PCI However,

those patients were recruited by the same investigators as

noted earlier (Staat et al., 2005) and around the same time.

The authors have not disclosed the clopidogrel usage in that

cohort

These observations on the effect of clopidogrel and other

P2Y12 receptor inhibitors are more than academic Use of

these agents in patients with AMI is now standard of care and

their dosing has been optimized so that most of today’s

patients may already be postconditioned at the time of PTCA

Hence, any additional intervention using the same protective

mechanism as the P2Y12receptor inhibitor would add little to

the protection This problem is compounded by many recent

clinical trials that have led to incremental improvements in

antiplatelet treatment New blockers have greatly shortened

absorption times and their dosing is being optimized so that

eventually all patients presenting with AMI are likely to be

optimally postconditioned To obtain additional

cardiopro-tection, an adjunct intervention must have a different

mechanism of action

Additional cardioprotection in the presence of

an antiplatelet drug

We have found that some protective interventions can be

combined to produce more potent protection Unlike IPoC

and cangrelor, mild hypothermia (Miki et al., 1998b; Tissier

et al., 2007b) and cariporide (Miura et al., 1997) are most

protective when applied during ischaemia rather than

reper-fusion Thus, protection from IPC which protects against a

reperfusion injury could be added to that of cooling which

protects against an ischaemic injury (Miki et al., 1998b)

Simi-larly, the protective effect of the pharmacological

postcondi-tioning agent AMP579 which protects against a reperfusion

injury (Xu et al., 2003) could be added to that of cariporide

that protects against an ischaemic injury (Xu et al., 2002).

We tested whether any interventions could be additive

with cangrelor treatment (Yang et al., 2013a) In rats,

perito-neal lavage with ice-cold saline 10 min before coronary

occlu-sion lowered blood temperature to 32–33°C and decreased

infarction to 25% of the risk zone, equivalent to that seenwith cangrelor treatment just before reperfusion The twointerventions together halved infarction to 14% of myocar-dium at risk Cariporide’s protective effect (27% infarction) iscomparable with cangrelor’s The combination of cangrelorand cariporide nearly halved infarction to 16% of the riskzone When all three interventions were combined, infarc-tion again halved to only 6% of the risk zone Obviously,treatment during the ischaemic period is logistically problem-atic However, most patients spend an extended period oftime with healthcare professionals before recanalization hasbeen accomplished during which time these interventionscould be effectively implemented

We suggest that future animal studies of cardioprotectiveinterventions be conducted on a background of a P2Y12

inhibitor to provide a more clinically relevant model Unless

an agent can provide additive protection in that model, itwould be of little clinical value Although we have tested IPCand IPoC combined with the P2Y12inhibitor cangrelor andhave found no additional protection, it is unknown whetherinterventions such as remote conditioning (Lim andHausenloy, 2012) or promoters of autophagy (Sala-Mercado

et al., 2010) whose mechanisms are less well understood

might have additive effects

Concluding remarks

Our 43-year journey has brought us to this point where wehave a good understanding of the type of intervention thatmust be introduced to spare ischaemic myocardium Nowthat conditioning’s protection is being applied to patientsroutinely with the antiplatelet drugs, we should look else-where for the next generation of cardioprotective drugs Wepropose that an intervention not based on the signalling ofIPC or IPoC would be most likely to add protection to thatalready resulting from treatment with standard antiplateletagents Thus, IPoC or even cyclosporin which prevents for-mation of mPTP would be expected to have only small, if any,effect in this setting Infarct size reduction clearly reducesmortality and morbidity in AMI as clinical trials with reper-fusion therapy and P2Y12inhibitors have proven Yet furtherprotection against infarction is still indicated as AMI contin-ues to be a deadly and debilitating disease The preclinicalstudies completed by many investigators have established thefoundation for cardioprotective strategies and we must con-tinue to search for new strategies to preserve myocardiumfrom a transient ischaemic insult In the past, the improve-ments in outcome in AMI have been incremental rather thanrevolutionary By using relevant animal models, we can hope-fully identify candidates for future clinical testing andsomeday make myocardial infarction a mere historicalmedical footnote

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H579–H588

Themed Section: Conditioning the Heart – Pathways to Translation

REVIEW

Postconditioning signalling

in the heart: mechanisms

and translatability

Justin S Bice and Gary F Baxter

School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK

Correspondence

Professor Gary F Baxter, School ofPharmacy and PharmaceuticalSciences, Cardiff University, KingEdward VII Avenue, CardiffCF10 3NB, UK E-mail:

baxtergf@cardiff.ac.uk -

identified potential pharmacological targets for limitation of reperfusion injury These include ligands for membrane-associatedreceptors, activators of phosphokinase survival signalling pathways and inhibitors of the mitochondrial permeability transitionpore In experimental models, numerous agents that target these mechanisms have shown promise as postconditioningmimetics Nevertheless, clinical studies of ischaemic postconditioning and pharmacological postconditioning mimetics areequivocal The majority of experimental research is conducted in animal models which do not fully portray the complexity ofrisk factors and comorbidities with which patients present and which we now know modify the signalling pathways recruited

in postconditioning Cohort size and power, patient selection, and deficiencies in clinical infarct size estimation may allrepresent major obstacles to assessing the therapeutic efficacy of postconditioning Furthermore, chronic treatment of thesepatients with drugs like ACE inhibitors, statins and nitrates may modify signalling, inhibiting the protective effect of

postconditioning mimetics, or conversely induce a maximally protected state wherein no further benefit can be demonstrated

Arguably, successful translation of postconditioning cannot occur until all of these issues are addressed, that is, experimental

investigation requires more complex models that better reflect the clinical setting, while clinical investigation requires biggertrials with appropriate patient selection and standardization of clinical infarct size measurements

BJP British Journal of

Development of the postconditioning

paradigm for cardioprotection

Death due to acute myocardial infarction (AMI) has declined

steadily in the economically developed countries during the

last 50 years Since the 1980s, the development of reperfusion

therapies as the ‘standard of care’ for AMI has contributed

markedly to the decline in early mortality However, while

case fatality rate has declined, there is evidence of an

increas-ing incidence of chronic heart failure in AMI survivors It is

likely that infarct size is a major determinant of myocardial

remodelling processes that predispose patients to the

subse-quent development of heart failure Thus, prompt

reperfu-sion is needed to effectively limit the development of

ischaemic necrosis during AMI, but it seems plausible that

further limitation of infarction is desirable to reduce

long-term morbidity and mortality due to heart failure The

iden-tification of potential adjunctive infarct-limiting treatments

has been a goal of experimental cardioprotection research for

several decades A vast array of pharmacological and other

interventions have been described A review of all of these is

beyond the scope of this paper, but we wish to highlight here

three pivotal conceptual developments that have emerged

over several decades and converged to provide a new

cardio-protection paradigm around 2005

1 A mechanism of tissue injury specifically associated with

reperfusion, termed ‘lethal reperfusion injury’, was

pro-posed as long ago as the late 1970s (Ashraf et al., 1978;

Hearse et al., 1978) This concept implied the rapid

irre-versible injury, or accelerated death, of cells still viable at

the end of an ischaemic period as a result of the suddenreintroduction of oxygen to ischaemic tissue Thisreperfusion-associated cell death would be expected tocontribute to final infarct size in reperfused AMI For twodecades, the concept of lethal reperfusion injury proved to

be controversial The proposed molecular and cellularmechanisms of lethality were diverse and poorly defined.Most importantly, experimental pharmacological inter-ventions specifically targeted at reperfusion were not con-sistent in their infarct-limiting ability However, from themid-1990s, there was increasing evidence that apoptoticsignals are activated during early reperfusion and thatreperfused myocardium displays hallmarks of apoptosis.Although the quantitative contribution of apoptosis toinfarct size is likely to be small, experimental activation ofanti-apoptotic survival signals and inhibition of caspaseswere found to limit infarct size in experimental models.This work led to the development by Yellon and colleagues

of a general hypothesis that attenuation of lethal sion injury and limitation of infarct size could be induced

reperfu-by activating anti-apoptotic survival signals termedthe ‘reperfusion injury salvage kinase’ (RISK) pathway(Hausenloy and Yellon, 2004)

2 In 1986, Murry et al (1986) made the experimental

obser-vation that brief periods of ischaemia preceding AMI led to

an acute adaptation of myocardium that limited infarctsize This phenomenon was termed ischaemic precondi-tioning Intensive research throughout the 1990s revealedthat ischaemic preconditioning is associated with therecruitment of a number of autacoid-stimulated signaltransduction mechanisms that enhance the tolerance ofmyocardium to ischaemia-reperfusion insult, and thereby

Catalytic receptorsc Soluble GC

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

BJP J S Bice and G F Baxter

limit infarct size The first autacoid to be identified in

relation to the preconditioning mechanism was

adeno-sine This factor had been investigated extensively before

the discovery of preconditioning in relation to nucleotide

metabolism in ischaemia (Berne and Rubio, 1974; Berne,

1980) Indeed, ATP catabolism had been an area of active

investigation in the laboratory of Reimer and Jennings for

many years (Reimer et al., 1986) and led directly to the

experimental protocol that identified preconditioning

(Murry et al., 1986) Subsequently throughout the 1990s,

several other autacoid factors and numerous intracellular

signal transduction mechanisms were identified, all

pre-sumed only to be effective if activated before the onset of

AMI While preconditioning mechanisms induce a marked

and very reproducible infarct-limiting effect, the clinical

utility of therapies based on these mechanisms is

extremely limited since pre-ischaemic treatment is not a

possibility for the majority of AMI patients in whom

coro-nary thrombosis is sudden and unexpected

3 In 2003, Vinten-Johansen and colleagues reported that

intermittent repetitive re-occlusion of the infarct-related

coronary artery during the early moments of reperfusion

in an experimental model of AMI was able to limit infarct

size as effectively as ischaemic preconditioning (Zhao

et al., 2003) This reperfusion-specific intervention is

termed ischaemic postconditioning Subsequent early

research on postconditioning was remarkable for several

reasons Firstly, postconditioning confirmed that lethal

reperfusion injury contributes significantly to final infarct

size Secondly, both ischaemic preconditioning and

ischaemic postconditioning were shown to involve the

activation, during reperfusion, of the RISK pathway

iden-tified a few years previously (Kin et al., 2004) Clearly, the

temporal characteristics of postconditioning highlight the

relative importance of reperfusion injury in AMI, but have

no effect on ischaemic injury even though ischaemia is the

sine qua non of AMI.

Introduction of the postconditioning paradigm for

car-dioprotection has attracted huge interest as a possible

thera-peutic intervention at reperfusion to limit the injurious

combined effect of ischaemia and reperfusion In this respect,

intervention at reperfusion with conditioning protocols or

with pharmacological agents that replicate conditioning

mechanisms can truly be said to represent a paradigm shift in

the field

Characteristics of postconditioning

Interventions applied in the early reperfusion period to

augment tissue salvage, beyond that achieved by reperfusion

alone, are now often described as postconditioning

treat-ments Such interventions may take several forms and it is

important to distinguish between them Here we provide a

brief overview of these interventions and their major

charac-teristics: for further discussion, the reader is referred to more

detailed reviews elsewhere (Burley and Baxter, 2009; Ovize

et al., 2010; Shi and Vinten-Johansen, 2012; Hausenloy,

2013)

Myocardial ischaemic postconditioning

Postconditioning to limit infarct size was first formallydescribed and characterized in the open-chest dog by Zhao

et al (2003) This form of postconditioning is referred to as

myocardial ischaemic postconditioning, classical tioning or mechanical postconditioning It has beendescribed in several other experimental species (mouse, rat,

postcondi-rabbit and pig) in vivo (Yang et al., 2004; Schwartz and Lagranha, 2006; Tang et al., 2006; Gomez et al., 2008), in isolated rodent heart preparations (Tsang et al., 2004; Heusch et al., 2006), in humans and in isolated human myo- cardium (Sivaraman et al., 2007) The major characteristic of

the intervention is that brief (typically 10–30 s), repetitiveperiods (3–10 cycles) of ischaemia, interspersed with brief(10–30 s) periods of reperfusion, are achieved by physicalocclusion and reperfusion of the infarct-related coronaryartery immediately following the index ischaemic event (seeFigure 1) Most studies suggest that the timing of the inter-vention is critical to the outcome, a reduction in infarctsize A delay of more than 1 min in instituting the firstre-occlusion of the coronary artery was associated with a loss

of protection (Skyschally et al., 2009) This concurs with the

prevailing view that lethal reperfusion injury, associatedwith opening of the mitochondrial permeability transitionpore (MPTP), occurs within the first few minutes of reperfu-

sion (Griffiths and Halestrap, 1995; Di Lisa et al., 2001).

However, there is some evidence from the mouse heart gesting that myocardial ischaemic postconditioning canlimit infarct size if instituted even 30 min after reperfusion

sug-(Roubille et al., 2011) This effect has been termed ‘delayed’

ischaemic postconditioning Whether this is a phenomenonthat is generally applicable to other species, includinghumans, is not clear However, it has been suggested thatthese observations support the concept of a gradually evolv-ing ‘wavefront of reperfusion injury’, susceptible to laterintervention

Although ischaemic postconditioning has been reported

in every animal species examined, there is considerable ations in the extent of infarct limitation between species andlaboratories Murine models of postconditioning typicallydisplay 30% relative reduction in infarct size whereas in largermodels, such as rabbit and canine hearts, infarct size limita-

vari-tion is around 50% (Vinten-Johansen et al., 2011) The

post-conditioning protocols used and the duration of the period ofischaemia employed in these experimental models vary con-siderably Some data suggest that the threshold for ischaemicpostconditioning rises as the ischaemic duration increases.There have been some studies showing that ischaemic post-conditioning is unable to limit infarct size (Schwartz and

Lagranha, 2006; Dow and Kloner, 2007; Hale et al., 2008).

Typically, those studies that failed to show infarct limitationfollowing postconditioning used a shorter period of ischae-mia, and for larger animals, shorter postconditioning cycles

It is clear that there is not one protocol that suits all modelsand differences in protocols may account for the varyingdegrees of protection

In addition to limiting infarct size, ischaemic tioning has been reported to limit the severity of other del-eterious consequences of reperfusion These include the

postcondi-development of arrhythmias in the rat heart (Dow et al.,

BJP

Myocardial postconditioning

2008), cardiomyocyte apoptosis and the extent of vascular

injury (Schwartz and Kloner, 2012)

Remote ischaemic postconditioning

Numerous experimental and clinical observations suggest

that intermittent ischaemia at the onset of myocardial

reper-fusion of tissues and organs remote from the heart can limit

myocardial infarct size (see Figure 1) This phenomenon,

called remote ischaemic postconditioning (or inter-organ

postconditioning), is the subject of a comprehensive review

elsewhere in this issue (Schmidt et al., 2014) The most

fre-quently applied remote ischaemic postconditioning

inter-vention in both experimental and clinical models is

intermittent limb ischaemia performed at the onset of

myo-cardial reperfusion (Kharbanda et al., 2001; Loukogeorgakis

et al., 2006) The potential utility of such a simple

interven-tion (e.g repeated inflainterven-tion of a blood pressure cuff) hasattracted considerable interest, further augmented by the rec-ognition that some benefit also accrues if the remote post-conditioning intervention is delayed by 30 min aftermyocardial reperfusion (‘delayed remote ischaemic postcon-ditioning’) The biological mechanisms of remote ischaemicpostconditioning are unclear, but there appears to be adependency on several interacting factors, including neuro-nal and humoral factors as well as transmission of unknown

factors via microvesicles (Giricz et al., 2014).

Figure 1

Schematic representation of myocardial postconditioning protocols and reported infarct limitation afforded by these interventions Indexischaemia, typically of 30 min duration, is followed by intermittent reperfusion-reocclusion of the coronary artery-ischaemic postconditioning.Similarly, when the reocclusion cycles are delayed by as little as 60 s, infarct limitation is no longer afforded Pharmacological postconditioningtypically involves the administration of a postconditioning mimetic during early reperfusion Remote postconditioning is afforded by occlusion-reperfusion cycles of an artery distal to the myocardium, typically a limb Modified reperfusion is initiated by gradual reperfusion of the occludedarea of the myocardium over several seconds Temporarily reducing the pH during the first minutes of reperfusion can also limit the infarct AcR,acidified reperfusion; BAY, cGMP elevating compounds; BNP, brain natriuretic peptide; GrR, gradual reperfusion; I/R, infarct to risk zone size; IPOC,ischaemic postconditioning; RPOC, remote postconditioning

BJP J S Bice and G F Baxter

Pharmacological postconditioning

The administration of pharmacological or other biologically

active agents during early reperfusion to effect

cardioprotec-tion is frequently termed pharmacological postcondicardioprotec-tioning

For clarity and precision, we believe that this term should be

reserved strictly for approaches that recruit or mimic the

established pathways associated with ischaemic

postcondi-tioning These approaches would include pharmacological

agonists for receptors that are known to participate in

ischae-mic postconditioning (e.g adenosine A2 receptor ligands or

kinin B2 receptor ligands) or activators of established signal

transduction mechanisms that are invovled in ischaemic

postconditioning (e.g statins and volatile anaesthetics

acti-vating the PI3K/Akt pathway or NO donors actiacti-vating the

cGMP/PKG pathway) It is usual for the administration of

such agents to be commenced shortly before reperfusion or

immediately after reperfusion onset Over many decades, a

wide variety of agents, unrelated directly to the mechanisms

of ischaemic postconditioning, have been reported to be

adjuncts to reperfusion These include calcium channel

blockers (Kloner and Przyklenk, 1991), magnesium salts

(Antman, 1995), caspase inhibitors (Mocanu et al., 2000) and

adrenoreceptor antagonists (Broadley and Penson, 2004)

Whether or not they are effective at limiting infarct size

during reperfusion, such pharmacological treatments should

not be described as postconditioning mimetics

Other modified reperfusion approaches

Several years before the formal description of ischaemic

post-conditioning, it was recognized that modified forms of

rep-erfusion could limit reprep-erfusion injury Most notable are

staged (gradual) reperfusion and acidic reperfusion (see

Figure 1) Several surgical studies in the 1980s showed that

gradual, rather than rapid, restoration of coronary blood flow

attenuated the development of reperfusion injuries

(arrhyth-mias and stunning) (Casale et al., 1984; Preuss et al., 1987).

This manoeuvre was later shown to limit infarct size (Sato

et al., 1997) Similarly, mild acidification of the blood or

crys-talloid perfusate during early reperfusion showed a similarly

protective effect (Inserte et al., 2008) Our understanding of

the molecular mechanisms of reperfusion injury has led to

speculation that both manoeuvres limit the opening of MPTP

during early reperfusion, a mechanism shared in common

with the various forms of postconditioning and discussed in

more detail below

Overview of mechanisms of ischaemic

postconditioning

The prevailing conceptual model (see Figure 2) within which

the majority of work on ischaemic postconditioning is

cur-rently undertaken postulates opening of MPTP during the

early minutes of reperfusion as being a critical event leading

to cell death In the post-conditioned myocardium, a number

of complex interlinked signalling pathways are activated by

intracellular factors and extracellular autacoids These

signal-ling pathways ultimately impinge on MPTP, reducing the

probability of its opening This mechanistic framework has

been built up through a considerable body of experimentalwork, including pharmacological and genetic targeting ofthese pathways, autacoids, and components of the MPTP Wewill now describe the key evidence supporting the currentmodel beginning with a discussion of the pivotal role ofMPTP

Mitochondrial permeability transition

Hunter and Haworth (1979) and Crompton et al (1987)

iden-tified the MPTP as a non-specific channel of defined diameterspanning the mitochondrial inner and outer membranes.More recent work by Halestrap and colleagues made the asso-ciation between reperfusion and the formation of this pore in

an active state They observed that the opening of the MPTP

is enhanced by adenine nucleotide depletion, as well aselevated phosphate and oxidative stress, which are biochemi-cal anomalies associated with ischaemia-reperfusion injury

(Halestrap et al., 1998) Opening of the MPTP permits the

passage of molecules up to 1.5 kDa and, with the entry intothe mitochondrial matrix of H+, results in the uncoupling ofoxidative phosphorylation, ATP depletion and the onset ofcell death by necrosis Work by Crompton and Costi (1988)and Griffiths and Halestrap (1993; 1995) provided direct evi-dence of MPTP opening at reperfusion, but not during ischae-mia Particular features of the intracellular environment inreperfusion appear to contribute to this activation of MPTP.They include oxidizing conditions associated with reactiveoxygen species (ROS) generation, intracellular Ca2+overloadand the reversal of intracellular acidosis as a result of H+

washout (Buja, 2013) It has been suggested that ischaemicpostconditioning and postconditioning mimetic stimuliattenuate the opening of the MPTP by reducing intracellular

Ca2+ overload and limiting ROS generation (Leung andHalestrap, 2008)

It remains unclear how Ca2+, ROS and H+interact with theMPTP, but it has been reported that binding of adeninenucleotide translocase ligands to cyclophilin-D (CYP-D), asubunit of the MPTP, increases sensitivity to Ca2 + Mice defi-cient in CYP-D could not be protected by an ischaemic post-

conditioning stimulus (Elrod et al., 2010) On the other hand,

cyclosporine-A (Cys-A) that inhibits MPTP opening bybinding to CYP-D limits infarct size when administered atreperfusion in most animal models tested and in humans

(Gedik et al., 2013).

Mitochondrial KATP(MKATP) channels offer another protective target through their ability to regulate ROS pro-duction and Ca2+overload Perfusion with the KATPchannelblocker 5-hydroxydecanoate abolished postconditioning pro-tection in the rat, whereas the KATPchannel opener diazoxide

cyto-significantly improved cardiac contractile activity (Jin et al.,

2012) It has been also suggested that intermittent targeting

of the MKATPchannel during reperfusion, mimicking ditioning, affords cardioprotection by ROS compartmentali-

postcon-zation (Penna et al., 2007) Interestingly, postconditioning

was blocked by administration of an antioxidant during early

reperfusion It is proposed that the early generation of ROS

may trigger MKATP channel opening and PKC activation,which are required for protection; this is supported by thefinding that a channel blocker and PKC inhibitor attenuated

this protection (Yang et al., 2004) However, the subsequent

BJP

Myocardial postconditioning

reduction in ROS may prevent MPTP opening (Clarke et al.,

2008)

Receptor-mediated mechanisms

The involvement of a number of extracellular autacoid

factors, elaborated or enhanced as a result of ischaemic

post-conditioning, has been explored extensively These factors

are the subject of a comprehensive discussion elsewhere in

this issue (Kleinbongard and Heusch, 2014) and the

inter-ested reader is referred there In brief , the autacoids that have

received most attention include adenosine, bradykinin and

opioid peptides Several studies have demonstrated that

ischaemic postconditioning delays the washout of

endog-enous adenosine and the subsequent receptor activation

affords protection (Kin et al., 2005) Different receptor

sub-types are implicated in different species with A2A and A3

receptors being important in rat (Kin et al., 2005), while A2B

receptor signalling is required in the post-conditioned rabbit

heart (Philipp et al., 2006) Bradykinin B2receptors have also

been implicated in postconditioning in the rat perfused with

the B2 receptor antagonist HOE140, which attenuated the

protection (Penna et al., 2007) Interestingly, perfusion of

bradykinin for 3 min during early reperfusion was unable to

afford protection, yet intermittent perfusion in a protocol

that matched mechanical postconditioning demonstratedcomparable infarct limitation This protocol was unsuccessfulwhen using adenosine The significance of the protectionafforded by bradykinin perfusion in this model remains to beelucidated

Most recently the opioid receptor has been reported

to play a part in postconditioning The opioid receptorantagonist naloxone abolished the protection afforded by

postconditioning alone (Zatta et al., 2008) Similar to the

observations made with adenosine, postconditioning appears

to prevent the washout of pro-enkephalin, suggesting abuild-up of endogenous opioid during postconditioning.These observations are supported by recent findings thatreport that κ opioid receptor activation limits infarct sizeduring early reperfusion, an effect that was blocked by

ERK1/2 inhibition (Kim et al., 2011).

Protein kinase mechanisms

The third and most complex element of the postconditioningmechanism is transduction of the extracellular signalsdescribed above to the mitochondria, leading to inhibition ofMPTP (see above) Signal transduction is via a number ofpathways involving protein kinase activation, often sequen-tially The discussion below focuses on the major kinases

Figure 2

Schematic representation of the identified components of postconditioning signalling in the myocardium Autacoid factors such as adenosine andopioids along with other extracellular factors initiate cytoprotective signalling through their sarcolemmal receptors Three distinct signallingpathways have been reported, including RISK, which involves PI3K/Akt and ERK and distal inhibition of GSK-3β cGMP/PKG signalling throughnatriuretic peptides and soluble GC activation is identified as an additional pathway distally targeting mitochondrial potassium channels SAFE,whose major components are TNF-α and JAK/STAT, has also been demonstrated to play a role in postconditioning Although described as distinctpathways, their cytoprotective actions are demonstrated to culminate on the mitochondria, specifically inhibition of the MPTP It remains to befully investigated as to what extent these pathways interact and co-localize NPR, natriuretic peptide receptor; PKG, cGMP-dependent PK; RTK,receptor tyrosine kinase; SERCA, sarcoplasmic/endoplasmic reticulum calcium ATPase; SR, sarcoplasmic reticulum

BJP J S Bice and G F Baxter

explored to date While these are grouped discretely for the

purposes of this discussion, it needs to be recognized that

considerable overlap and crosstalk are likely to exist between

these cascades

RISK pathway (PI3K/Akt and MEK/ERK)

The RISK pathway, initially described by Yellon’s group,

consists of two related signalling cassettes: PI3K/Akt and

MEK/ERK Both act in a number of biological systems as

anti-apoptotic pro-survival signals, classically activated by

extracellular ligands including peptide growth factors (Yellon

and Baxter, 1999) (see Figure 2) PI3K/Akt and MEK/ERK have

been repeatedly demonstrated as major players in mediating

the cardioprotective effects of postconditioning in rodent

models (Hausenloy, 2009) Tsang et al (2004) reported that

Akt was phosphorylated following six 10 s cycles of

reperfu-sion in the isolated perfused rat heart Furthermore,

endothe-lial NOS (eNOS) and p70s6K were also phosphorylated more

than in hearts that had undergone a standard reperfusion

protocol These findings were corroborated by observations

that the classical PI3K inhibitors wortmannin and LY294002

abolished the protective effect of postconditioning

Subse-quently, Yang et al (2004) reported the importance of MEK/

ERK signalling in an isolated rabbit heart model where

pharmacological inhibition of MEK/ERK activation abolished

the protection Of note, RISK signalling is implicated in the

cardioprotective effect of postconditioning in human atrial

muscle ex vivo (Sivaraman et al., 2007) Many

pharmacologi-cal mimetics of postconditioning have been shown to require

the participation of either PI3K/Akt or MEK/ERK or both

(Hausenloy, 2009)

GSK-3β

Inhibition of glycogen synthase kinase-3β (GSK-3β) is

associ-ated with cell survival and may be considered as a

down-stream component of RISK signalling Phosphorylation

inhibits GSK-3β activity and thereby inhibits MPTP activity

(Juhaszova et al., 2009) However, its relative importance has

been disputed in different models Wagner et al (2008)

reported that both GSK-3β and ERK phosphorylation are

sig-nificantly increased following postconditioning in rats These

observations were subsequently supported by further

bio-chemical analysis demonstrating increased GSK-3β

phospho-rylation following postconditioning in a rat global ischaemia

model In contrast, GSK-3β double knock-in mice could be

protected with a postconditioning stimulus in a global

ischaemia model (Nishino et al., 2008) Further evidence is

required to ascertain the precise contribution of GSK-3β and

how it may vary in different species

SAFE pathway (JAK/STAT3)

The survivor activating factor enhancement (SAFE) pathway

has been identified as an alternative cytoprotective pathway

to RISK that is triggered by TNF-α and JAK/STAT signalling

Lecour’s laboratory has reported that pharmacological

inhi-bition of the JAK/STAT pathway reverses the infarct limitation

afforded by postconditioning (Lacerda et al., 2009) They

also demonstrated that TNF-α signalling through TNFR2

also known as TNFRSF1B) and STAT3 is required to afford

protection The protection afforded was independent of PI3K/Akt and MEK/ERK signalling TNFR2 antibodies abolishedprotection afforded by postconditioning whereas TNFR1

knockout mice were still conditioned (Lacerda et al., 2009).

Protection observed with TNF-α was not present when theJAK/STAT3 inhibitor AG490 was administered at reperfusion

(Lecour et al., 2005) The upstream activators of the SAFE

pathway have attracted little attention to date, but it is gested that autacoids such as those found upstream of the

sug-RISK cascades could be involved (Hausenloy et al., 2013).

Distal to the SAFE pathway, it is suggested that signallingconverges on the mitochondria; however, whether the SAFEpathway converges on the same targets as RISK remains to beinvestigated thoroughly

cGMP/PKG pathway

Endogenous NO derived from eNOS is implicated in mic postconditioning in several animal models Pharmaco-logical inhibition of eNOS activity abolished the protective

ischae-effects of postconditioning (Tsang et al., 2004) Conversely,

many studies have demonstrated the cytoprotective effects ofadministering a NO donor in the first few minutes of reper-fusion, although this effect of NO donors is not consistently

seen (Bice et al., 2014a) NO activates soluble GC leading to

the generation of cGMP and subsequent activation of dependent PK (PKG) Several lines of evidence support theeffectiveness of this pathway as a cardioprotective cascade

cGMP-(Krieg et al., 2009; Bice et al., 2014b) In addition, cGMP/PKG

signalling through particulate GC targeting via natriureticpeptides has also been demonstrated to afford infarct limita-tion (Burley and Baxter, 2007) However, at present, it isunclear if the PKG pathway is an essential component ofischaemic postconditioning and if it sits alongside the PI3K/Akt pathway or is distal to it (see Figure 2)

Anti-apoptotic mechanisms

The relative contributions that apoptosis and necrosismake in reperfusion injury have long been debated Specifi-cally, the timing of apoptosis during the development ofmyocardial ischaemia/reperfusion injury remains unclear

Sun et al (2009) reported that postconditioning limited

myocardial apoptosis in rat neonatal cardiac myocytes Itwas reported that TUNEL staining was reduced comparedwith controls and that ROS generation and intracellular

calcium accumulation were reduced Cytochrome c and

caspase-3 have also been implicated in postconditioning

sig-nalling associated with a reduction in apoptosis Penna et al.

(2006) reported that these factors were reduced following

postconditioning in an ex vivo rat model, while increasing

the anti-apoptotic factor Bcl-2 Inflammatory mediatorsincluding cytokines have also been associated with apop-totic regulation Mechanical postconditioning has beenshown to decrease TNF-α and limit ROS formation duringearly reperfusion, resulting in attenuation of apoptosis (Kin

et al., 2008) Most recently, the apoptosis repressor with

caspase recruitment domain has been shown to decreasecaspase-3 activity and subsequent apoptosis in chickembryo myocytes following exposure to hydrogen peroxide

(Wu et al., 2013).

BJP

Myocardial postconditioning

Clinical studies of ischaemic and

pharmacological postconditioning

From the brief account above, it may be inferred that a

number of potential approaches exist for the development of

postconditioning as a clinical therapeutic intervention

Indeed, as proof of concept, Staat et al (2005) demonstrated

that a mechanical postconditioning algorithm could be

intro-duced in patients with AMI with significant reduction in a

surrogate marker of infarct size [serum creatine kinase (CK)

concentration] Over the last decade, further clinical trials of

ischaemic postconditioning have been conducted with

mixed outcomes In those studies that measured CK as an end

point, approximately half of them reported positive

out-comes (see Table 1) The remaining trials, all with small

cohort sizes, reported neutral or negative end points Most

recently, a comparatively large trial reported that four cycles

of 60 s reperfusion and re-occlusion failed to reduce peak

CK-MB (Hahn et al., 2013) (see Table 1) A number of

poten-tial explanations can be posited for the variability of clinical

studies of ischaemic postconditioning These include

varia-tions in postconditioning algorithms These issues are

dis-cussed comprehensively in a recent review (Ferdinandy et al.,

2014)

Pharmacological approaches to postconditioning have

been assessed in a number of clinical studies Here we

high-light some notable completed studies related to the

mecha-nisms outlined above

Adenosine

Adenosine was evaluated as an adjunct to clinical reperfusion

therapy before the formal identification of postconditioning

(Mahaffey et al., 1999) A reduction in infarct size of 33% was

demonstrated in patients receiving adenosine before

throm-bolysis and prompted a larger trial in which the primary end

points were development of congestive heart failure or 6

month mortality rates (Ross et al., 2005) The results of this

2118 patient trial were disappointing with no significant

improvement in primary outcomes There was, however, a

suggestion that in a subset of patients, infarct size was

reduced in patients who received the highest dose of

adeno-sine Furthermore, post hoc analysis suggested that benefit was

only observed in patients who received early adenosine

treat-ment (Kloner et al., 2006) Almost half of the patients in the

follow-up trial underwent angioplasty rather than

throm-bolysis which also needs to be considered

cGMP/PKG pathway

Most recently, the results of the NIAMI trial have been

pub-lished in which nitrite was administered before percutaneous

coronary intervention (PCI) as a source of exogenous NO

(Siddiqi et al., 2014) Extensive experimental studies have

demonstrated the protective effects of administering nitrate,

nitrite or NO donors before reperfusion Indeed, nitrite has

been shown to have vasorelaxant and anti-platelet properties

which may be enhanced under ischaemic conditions, but

these are actions unrelated to a postconditioning effect

(Rassaf et al., 2014) Post hoc analyses of patients who had

been undergoing chronic nitrate therapy were shown to have

fewer ST-elevated myocardial infarctions compared with

patients who were described as nitrate nạve (Ambrosio et al.,

2010) However, in the NIAMI trial, no reduction in infarctsize measured by cardiac magnetic resonance imaging wasreported in patients receiving sodium nitrite 5 min beforePCI

Targeting the cGMP pathway and the KATPchannel hasalso been explored in the clinical setting The large multicen-tre J-WIND trial treated patients with atrial natriureticpeptide after reperfusion treatment which showed approxi-mately 15% reduction in total CK release Patients treatedwith the KATPchannel opener nicorandil did not show any

significant reduction in total CK release (Kitakaze et al.,

2007)

MPTP inhibition

In a small pilot study, Cys-A limited infract size by 20%compared with controls when measured by MRI 5 days after

treatment (Piot et al., 2008) Furthermore, no adverse effects

of Cys-A were reported An ongoing multicentre trial(CIRCUS) is further investigating the potential of Cys-A as anadjunct to reperfusion, the primary end points being hospi-talization for heart failure and left ventricular remodelling at

1 year

Other pharmacological agents

In addition to exploring pharmacological postconditioningmimetics, other agents that may offer protection in the clini-cal reperfusion setting have been investigated Statins, βblockers, erythropoietin (EPO), glucagon-like peptide andglucose-insulin-potassium have all been utilized in clinicaltrials with varying outcomes Two small trials in which EPOwas administered prior to PCI reported conflicting outcomes

(Ferrario et al., 2011; Suh et al., 2011) Similar doses were

used; however, a 30% reduction in CK-MB was reported inone and no improvement was reported in the other Secondand third doses were, however, administered at 24 and 48 h

in the positive outcome trial The proposed mechanism ofaction for EPO protection is said to involve inhibition ofthe myocardial inflammatory response which may have adelayed component explaining the differences in clinicaloutcomes

The challenges and opportunities for successful translation

The picture obtained so far is that myocardial ischaemicpostconditioning has the potential to limit infarct size but is

of variable efficacy Clinical studies with pharmacologicalmimetic approaches (e.g adjuncts to PCI or thrombolysis forAMI) that target the postconditioning signalling pathwaysdescribed in experimental studies have not been overwhelm-ingly positive There are likely to be many reasons for theseinconsistent findings They include study design features (e.g.patient inclusion criteria, timing of drug administration);technical limitations to accurate end point assessment (e.g.normalized infarct size measurement in humans); andattenuation or the overwhelming of the postconditioningsignalling mechanisms in patients The latter potentially

BJP J S Bice and G F Baxter

Table 1

Clinical trials utilizing mechanical and pharmacological postconditioning in patients presenting with STEMI

flow velocity

in myocardial salvage

PI3K/Akt

within 15 min R

cGMP/PKG

pre-PCI

Mitochondria

size (CMR)

IPOC, ischaemic postconditioning; LVEF, left ventricular ejection fraction; MI, myocardial infarction; NS, not significant; R/I, cycle ofreperfusion and ischaemia; SPECT, single-photon emission computer tomography; STEMI, ST-elevated myocardial infarction; WMSI, wallmotion score index

BJP

Myocardial postconditioning

represents the greatest challenge for successful translation of

postconditioning into the therapeutic arena

The confounding effect of comorbidities

The majority of experimental studies of ischaemic

postcon-ditioning or pharmacological postconpostcon-ditioning mimetics

have been performed in healthy, juvenile male animals

These models are devoid of associated risk factors for

cardio-vascular disease and do not represent the comorbidities often

present in the clinical setting It is now clear that many of the

risk factors and comorbid conditions that contribute to or are

present in coronary artery disease (senescence, gender-related

hormonal background, dyslipidaemia, hypertension,

diabe-tes, etc.) modify the signalling pathways underpinning

post-conditioning (Downey and Cohen, 2009; Przyklenk, 2013;

Vander Heide and Steenbergen, 2013; Ferdinandy et al.,

2014) In experimental models that address these factors,

both ischaemic and pharmacological postconditioning

effects may be abolished or severely attenuated because of

biochemical perturbations brought about by these

condi-tions The worrying possibility that the majority of

experi-mental models have not predicted or do not resemble clinical

reality might be regarded by some as the killer blow for

successful development of clinical postconditioning and may

go some way to explaining the massive variability in clinical

trials to date However, we are not so pessimistic It seems

plausible that at least with some of these comorbidities,

post-conditioning is not completely abolished, but rather the

threshold for activation of the pathways is raised For

example, in experimental studies where protection by either

ischaemic postconditioning or Cys-A was abolished in

dia-betic hearts, the combination of both interventions restored

protection suggesting that the diabetic heart could be

pro-tected if an increased cardioprotective threshold could be met

(Badalzadeh et al., 2012) Moreover, in some cases, treatment

or resolution of the comorbidity restores the

postcondition-ing effect For example, in a rabbit model, postconditionpostcondition-ing

alone could not limit infarct size in high-cholesterol-fed

animals However, administration of pravastatin was able to

afford protection in these resistant animals, an effect that was

blocked by eNOS inhibition (Andreadou et al., 2012).

The confounding effect of current

drug therapies

Another intriguing possible explanation for variability in

clinical postconditioning studies is that many patients are in

fact already in a maximally conditioned state as a result of

their existing drug therapy Bell and Yellon (2014) have

recently proposed a ‘success hypothesis’ suggesting that

many, perhaps the majority of, patients presenting with acute

coronary syndromes are already conditioned by the

polyp-harmaceutical regimen of drugs that they are already taking

Statins, ACE inhibitors,β blockers and opioid analgesics are

all commonly prescribed to these patients and indeed

have all been shown to be cardioprotective or to have

conditioning-like properties in the experimental setting On

the other hand, these drugs have been found to inhibit

con-ditioning mechanisms in some studies The term ‘hidden

cardiotoxicity’ has been proposed which suggests that some

of the adjunct therapies used may increase the threshold for

cardioprotection (Ferdinandy et al., 2014).

Clinical trial design and translating postconditioning

The disparity between the experimental studies and the cal trial data obtained so far suggests that translation – both

clini-from bench to bedside and vice versa – needs to be improved.

As identified above, experimental study design needs to berefined for further mechanistic studies to represent better theclinical setting At the very least, experimental models inwhich comorbidities can be simulated should be used follow-ing initial mechanistic studies It is clear that we need to focus

on building on the well-documented signalling cascades andthe spatial and temporal modifications to signalling in dis-eased states

To date, the majority of clinical trials assessing cological postconditioning mimetics have been unsuccessful

pharma-or of only modest benefit (see Table 1) But their limitedsuccess may be explained in two ways Firstly, the design ofthe preclinical animal experiments may fail to resemble thecomplexities of the clinical situation and this leads to inap-propriate target selection Secondly, the design of a clinicaltrial needs to account for the massive heterogeneity of thepatient population and recognise the currently limited ability

to quantify tissue salvage or measure infarct size standardized

to risk zone size accurately and reliably Unlike laboratoryspecies, the clinical population presenting with AMI is aheterogeneous mix of high-risk and low-risk patients, thosewith large infarcts and those with small infarcts Unlike thelaboratory experiment, the ischaemic risk zone size, the dura-tion of the ischaemic episode and the speed of successfulreperfusion are highly variable in human AMI Perhaps mostimportantly, the high degree of standardization of infarct sizemeasurement required in the experimental laboratory iseffectively unachievable in the clinical setting with presentlyavailable methods

Thus, it seems unlikely that we will achieve a tioning intervention that guarantees benefit for all Muchmore likely is that an agent that is safe and easy to administer

postcondi-as a single dose – probably a repurposed drug such postcondi-as Cys-A –could be given to all AMI patients undergoing reperfusionwith the expectation that a proportion might benefit Giventhe very large number of patients undergoing reperfusiontherapy, the global benefit of such an approach could belarge

Conflict of interest

There is no conflict of interest to disclose

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Themed Section: Conditioning the Heart – Pathways to Translation

Michael Rahbek Schmidt1, Andrew Redington2and Hans Erik Bøtker1

1Department of Cardiology, Aarhus University Hospital Skejby, Aarhus N, Denmark, and

2Division of Cardiology, Hospital for Sick Children, Toronto, ON, Canada

Correspondence

Hans Erik Bøtker, Department ofCardiology, Aarhus UniversityHospital Skejby,

Brendstrupgaardsvej 100,DK-8200 Aarhus N, Denmark.E-mail: heb@dadlnet.dk -

short-lasting-induced ischaemia of the heart itself or a remote tissue Remote ischaemic conditioning (RIC) in particular hasbeen utilized in a number of clinical settings with promising results However, while many novel ‘downstream’ mechanisms ofRIC have been discovered, translation to pharmacological conditioning has not yet been convincingly demonstrated in clinicalstudies One explanation for this apparent failure may be that most pharmacological approaches mimic a single instrument in

a complex orchestra activated by mechanical conditioning Recent studies, however, provide important insights into upstreamevents occurring in RIC, which may allow for development of drugs activating more complex systems of biological organprotection With this review, we will systematically examine the first generation of pharmacological cardioprotection studiesand then provide a summary of the recent discoveries in basic science that could illuminate the path towards more advancedapproaches in the next generation of pharmacological agents that may work by reproducing the diverse effects of RIC,thereby providing protection against IR injury

From acute events such as stroke and acute myocardial

infarction (MI) to predictable circumstances such as elective

surgery and angioplasty, the injury caused by ischaemia and

reperfusion is a leading cause of death and disability (Murray

and Lopez, 1997; Wang et al., 2012) Since 1990, more people

have died from coronary heart disease than any other cause

of death (Lloyd-Jones et al., 2009; 2010) Ischaemia–

reperfusion (IR) syndromes remain a major clinical challenge

worldwide

In acute coronary events, early and successful restoration

of myocardial reperfusion following an ischaemic event is the

most effective strategy to reduce final infarct size and improve

clinical outcome, but reperfusion may induce further

myo-cardial damage itself, so-called reperfusion injury (Murry

et al., 1986) Although the process of myocardial reperfusion

continues to improve with more timely and effective

coro-nary intervention and antiplatelet and antithrombotic agents

for maintaining the patency of the infarct-related artery, the

development of effective drugs to treat the detrimental effects

of reperfusion injury itself has proven to be a challenge

Indeed, several pharmacological strategies showing

convinc-ing effects in animal models of IR injury have failed to

trans-late to clinical benefit

Consequently, as the first generation of pharmacological

conditioning studies {including large clinical trials such

as AMISTAD-II (adenosine), APEX-MI (pexelizumab) and

CREATE-ECLA [glucose-insulin-potassium (GIK) infusion]}

(see Table 1) failed to show convincing effects, mechanical

ischaemic conditioning strategies have dominated the more

recent clinical trials

Since its conceptual demonstration in 1986, local

ischae-mic preconditioning of the heart (and subsequently other

organs) achieved by intermittent sub-lethal periods of mia prior to a longer lasting ischaemic insult has evolved intothe more clinically applicable methods of local ischaemic

ischae-postconditioning and remote ischaemic conditioning (RIC)

(see below), both of which have shown promising results in

clinical trials (Staat et al., 2005; Hoole et al., 2009; Botker

et al., 2010; Lonborg et al., 2010; Thielmann et al., 2010;

2013; Davies et al., 2013; Sloth et al., 2014) The increasing

insight into the mechanisms and pathways of ischaemicconditioning may pave the road for development of a newgeneration of cardioprotective drugs closely mimickingthe powerful inherent protection afforded by ischaemicconditioning

This review will focus upon novel advances in our standing of the mechanisms involved in RIC and thescope for potential development of novel pharmacologicalapproaches

under-Remote ischaemic conditioning

‘Remote ischaemic conditioning’ (RIC), induced by repeatedshort-lasting ischaemia in a distant tissue – largely achieved

by intermittent interruption of circulation in a limb – hasrecently emerged as a promising adjunctive therapy to avoidorgan damage, thereby improving the outcomes of well-established therapies From the site of the remote stimulus,

through humoral (Shimizu et al., 2009) and neuronal (Loukogeorgakis et al., 2005; Lim et al., 2010) pathways, RIC

activates several protective mechanisms in the target organsimilar to those activated by local preconditioning Further-more, RIC modifies the systemic inflammatory response

(Konstantinov et al., 2004; Shimizu et al., 2010), prevents endothelial dysfunction (Kharbanda et al., 2002) and platelet

Tables of Links

TARGETS

kinase 3β

Nuclear receptorsb JAK

NO, nitric oxideRapamycin (sirolimus)Spironolactone

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://

www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are

Alexander et al., 2013a,b,c,d,e)