ORIGINAL CONTRIBUTION

Remote ischaemic preconditioning involves signalling throughthe SDF-1a/CXCR4 signalling axis

Sean M. Davidson • Pradeep Selvaraj •

David He • Claire Boi-Doku • Robert L. Yellon •

Jose M. Vicencio • Derek M. Yellon

Received: 26 April 2013 / Revised: 26 June 2013 / Accepted: 29 July 2013

� Springer-Verlag Berlin Heidelberg 2013

Abstract Ischaemic preconditioning is one of the most

potent experimental modalities known to decrease infarct

size after ischaemia and reperfusion. Much interest has

been stimulated by the phenomenon of remote ischaemic

conditioning (RIC), in which the preconditioning stimulus

is applied to a limb remote from the heart to stimulate

cardioprotection via an unidentified humoral factor,

believed to be a protein between 3.5 and 15 kDa. Stromal

cell-derived factor-1 (SDF-1a or CXCL12) is a chemokine

of 10 kDa that is induced by hypoxia and recruits stem

cells, but also exerts direct, acute, cardioprotection via its

receptor, CXCR4. The serum dipeptidase DPPIV cleaves

and inactivates SDF-1a. We measured SDF-1a in rat

plasma and found it was significantly increased by RIC.

DPPIV activity was unchanged after RIC, suggesting that

increased synthesis or release or SDF-1a caused the

increase in plasma levels. AMD3100, a highly specific

inhibitor of CXCR4, was used to investigate the hypothesis

that SDF-1a is involved in RIC. RIC in rats, which

decreased infarct size from 53 ± 3 % to 27 ± 3 % (n = 6,

P \ 0.05), was blocked in rats treated with AMD3100

(40 ± 4 %). RIC also improved functional recovery of

cardiac papillary muscle, and this, too, was blocked by

AMD3100. Direct application of SDF-1a was confirmed to

be protective in this model and was blocked by AMD3100.

RIC stimulates SDF-1a release, and this 10-kDa peptide

appears to be required for the mechanism of RIC.

Keywords Ischaemia � Reperfusion � AMD3100 �Remote preconditioning � SDF-1a

Abbreviations

RIC Remote ischaemic preconditioning

SDF-1a Stromal cell-derived factor-1 alpha

CXCR C-X-C chemokine receptor

DPPIV Dipeptidase IV

Introduction

Ischaemic heart disease is a major cause of morbidity and

mortality throughout the world. Acute obstruction of an

artery can cause severe ischaemia followed by myocardial

infarction. Reperfusion via thrombolysis or percutaneous

coronary angioplasty (PCI) is the mainstay of treatment

and is necessary to limit the size of the evolving myocar-

dial infarction, but paradoxically itself causes injury,

termed ‘‘reperfusion injury’’ [45]. It has been known for

some 20 years that ischaemic preconditioning (IPC)—a

series of brief episodes of sub-lethal ischaemic and reper-

fusion—can protect against reperfusion injury [45]. A large

body of work has established that IPC is mediated by

protein kinases such as PI3 kinase, ERK/MAPK, PKC and

JAK/STAT [14]. These kinases act on a final common

pathway that prevents opening of the mitochondrial per-

meability transition pore (mPTP), thereby preserving

mitochondria and increasing cardiomyocyte survival [9,

14]. However, application of IPC directly to the heart is

impractical as a potential prophylactic treatment. As such

the discovery that a preconditioning protocol is effective

even when applied to an organ or tissue remote from the

heart has aroused a great deal of interest [16, 28]. Remote

ischaemic conditioning (RIC) can be induced non-

S. M. Davidson � P. Selvaraj � D. He � C. Boi-Doku �R. L. Yellon � J. M. Vicencio � D. M. Yellon (&)

The Hatter Cardiovascular Institute, University College London,

67 Chenies Mews, London WC1E 6HX, UK

e-mail: d.yellon@ucl.ac.uk

123

Basic Res Cardiol (2013) 108:377

DOI 10.1007/s00395-013-0377-6

invasively by repeatedly tying and releasing a ligature, or

by inflating and deflating a blood pressure cuff on a limb

[28]. We and others have demonstrated that RIC using such

a protocol can reduce myocardial injury in animals sub-

jected to I/R protocols and in patients undergoing cardiac

surgery [8, 15, 43], elective PCI [20], or in AMI patients

undergoing angioplasty [3, 18]. Although RIC is a clini-

cally amenable non-invasive, cost effective therapeutic

intervention that has been shown to reduce acute myocar-

dial injury [17], the mechanism by which it protects the

heart is as yet unknown. Studies have suggested that RIC

involves the release of a humoral factor(s) (probably a

peptide[3.5 kDa [37] and\15 kDa [38]), which is carried

by the blood from the limb to the remote target organ or

tissue [16].

Stromal cell derived factor-1a (SDF-1a) is CXC che-

mokine involved in the trafficking of hematopoietic and

lymphopoietic cells and the homing of stem cell progeni-

tors. Many studies have investigated its potential to recruit

stem cells to the injured heart in order to aid tissue

regeneration and healing [7, 27, 35, 42, 47]. In addition to

these chronic effects, several studies have demonstrated

that SDF-1a is also able to directly protect the isolated

heart from acute injury, by binding to its receptor CXCR4

[21, 22, 40]. The requirement for receptor binding was

demonstrated by the use of the specific inhibitor,

AMD3100. AMD3100 is regarded as being extremely

specific for CXCR4 [10] and does not interact with a

number of related chemokine receptors (CXCR1-3 or

CXCR1-9) [13].

Intriguingly, SDF-1a levels increase in response to

hypoxia [6]. Furthermore, studies have demonstrated that

the SDF-1a-CXCR4 signalling axis regulates diverse cell

functions via a Ga1-dependent mechanism and activation

of PI3 kinase, MAPK, PKC and JAK STAT signalling [21,

22, 35]—the same kinases known to be involved in the

RISK pathway [14], and known to be required for RIC [4,

19, 38, 41]. This led us to investigate whether SDF-1alevels increase after RIC, and whether inhibition of the

CXCR4 receptor with AMD3100 would prevent RIC.

Methods

Animal experiments

All work was carried out in accordance with the Guidelines

on the operation of Animals (Scientific Procedures) Act,

published by the UK home office in 1986. Male Sprague–

Dawley rats (300–400 g body weight) were obtained from

a breeding colony maintained at University College Lon-

don and randomly assigned to the control and treatment

groups. Rats were anaesthetized with sodium

pentobarbitone (60 mg/kg i.p.) and given heparin sodium

(300 IU). In order to exclude the effect of circulating cells

in the reduction of injury after ischaemia and reperfusion

(IR), RIC was first induced in anaesthetized rats. The hearts

were then surgically removed, cannulated via the aorta, and

placed on a Langendorff apparatus. RIC was administered

to anaesthetized animals placed on a warm mat, by tight-

ening of a ligature around one hind limb for 3 cycles of

5 min followed by release for 5 min. Complete cessation of

blood flow in the limb was verified by the use of Doppler

measurement. Control animals were subject to a sham

procedure for the same time-period, but without tightening

of the ligature until the end of the experiment, when the

ligature was tightened and Evans blue injected into the

heart to visualize the area at risk and confirm the correct

location of the suture. 10 lg/kg AMD3100 or vehicle

(DMSO) was delivered by i.p. injection 10 min before

RIC. Rats were randomized between control, RIC, AMD,

and AMD ? RIC groups, each administered in vivo as

described above, following which hearts in all four groups

were subject to IR in vitro on the Langendorff apparatus as

follows. Hearts were quickly removed via thoracotomy and

perfused retrogradely with modified Krebs–Henseleit buf-

fer (in mM: NaCl 118.5, NaHCO3 25.0, KCl 4.8, MgSO4

1.2, KH2PO4 1.2, CaCl2 1.7 and glucose 11.0), on a stan-

dard Langendorff apparatus (ADinstruments) at constant

pressure of 100 mmHg. All solutions were gassed with

95 % O2/5 % CO2 with pH maintained between 7.35 and

7.45 at 37 �C and temperature between 36.5 and 37.5 �C.

A latex, isovolumic balloon was introduced into the left

ventricle through the left atrial appendage and inflated to

give a pre-load of 8–10 mm Hg. Left ventricular developed

pressure, heart rate and coronary flow were recorded at the

middle of each time-period, i.e. stabilization, ischaemia,

and reperfusion (Fig. 1). As expected, coronary flow, heart

rate and LVDP, a measure of cardiac function, decreased

during ischaemia, and recovered during reperfusion,

though there were no significant differences between the

treatment groups (Fig. 1). After 40 min stabilization,

regional ischaemia was induced for 35 min by tightening a

suture around the left anterior descending artery (LAD)

followed by 1 h of reperfusion. At the end of the experi-

ment, the suture around the LAD was tightened and Evans

blue dye injected to demarcate the risk area. Hearts were

stained with triphenyl tetrazolium chloride, sectioned and

infarct size analysis was carried out using Image-J soft-

ware. The results are expressed as the percentage of the left

ventricle that was at risk (AAR), and percentage of area at

risk that was infarcted.

For the functional experiments hearts from anaesthe-

tized rats were removed and placed in ice-cold buffer and

the papillary muscle was carefully dissected. The muscle

was suspended in a heated organ bath under superfusion

Page 2 of 10 Basic Res Cardiol (2013) 108:377

123

with a modified Tyrode’s buffer [39] oxygenated with

95 % O2/5 % CO2 at 37 �C and one end of the muscle was

attached to a force transducer. The muscle was initially

stretched until 1 N, and then electrically stimulated to

contract at 1 Hz for 90 min to stabilize. They were then

subject to 30-min simulated ischaemia by superfusion with

glucose-free, hypoxic buffer gassed with 95 % N2/5 %

CO2, following which the muscle underwent reoxygenation

for 120 min with normoxic buffer, to simulate reperfusion,

with field stimulation at 1 Hz [39]. AMD3100 (5 lg/ml) or

vehicle (DMSO) was delivered for 15 min prior to

hypoxia. SDF-1a (25 ng/ml dissolved in buffer) was

delivered for 10 min prior to hypoxia. This value is higher

than routinely measured in plasma, but was selected based

on previous publications in isolated hearts [22], and based

on the assumption that a higher concentration may be

required to allow sufficient activation in a non-perfused

muscle. Normoxic control samples were perfused with

normoxic buffer throughout the procedure.

DPPIV assay

DPPIV activity was measured using the DPPIV-GLOTM

protease assay (Promega) according to manufacturer’s

protocol. After control or RIC procedure as above, rats

were exsanguinated by ventricular puncture into citrated

syringes, followed by centrifugation at 1,600g for 20 min

followed by 10,000g for 30 min to obtain platelet-free

plasma. Plasma samples were diluted 1:100 in assay buffer

(80 mM MgCl2, 140 mM NaCl, 1 % BSA in 50 mM

Hepes pH 7.8), added to an equal volume of DPPIV-

GLOTM reagent, gently mixed and incubated at RT for

30 min. Samples were compared to a standard curve of

DPPIV enzyme (r2 [ 0.99). Luminescence was measured

on a 96-well plate reader (BMGlabtech). As a negative

control, the DPPIV inhibitor sitagliptin was administered to

one group of animals by oral gavage 10 mg/kg 24 h before

exsanguination.

SDF-1a ELISA

To determine whether RIC increased plasma SDF-1a lev-

els, ELISA was used to measure total SDF-1a in plasma

samples from control rats and from rats immediately after

completion of the RIC procedure. Plasma was collected on

ice in heparinized tubes containing 5 ll of 50 mM sitag-

liptin to inhibit DPPIV and prevent proteolysis of SDF-1aduring isolation, then centrifuged at 1,0009g for 20 min

then 10,0009g for 30 min to remove platelets. SDF-1a was

assayed by Quantikine� sandwich ELISA (R&D systems)

and compared to a recombinant SDF-1a standard according

to the manufacturer’s protocol.

Western blot analysis

The expression of CXCR4 (Abcam ab2074, Cambridge,

UK) was analysed in LV rat myocardium and primary adult

rat cardiomyocytes by Western blot analysis. Band inten-

sity was determined by computerized densitometry using

NIH Image J 1.63 software and expressed as arbitrary units

after normalization to tubulin levels (Abcam, Cambridge,

UK).

Fig. 1 Functional data from Langendorff perfused hearts. a Heart

rate, b coronary flow, c LVDP. Though coronary flow and LVDP

were the highest during stabilization but decreased during simulated

ischaemia and recovered somewhat during reoxygenation; as

expected, there were no significant differences between the treatment

groups (Control, vehicle; RIC, remote conditioning; AMD,

AMD3100; R ? A, RIC ? AMD3100)

Basic Res Cardiol (2013) 108:377 Page 3 of 10

123

Statistics

All values are expressed ± SEM. Pairwise comparisons

were made by Students T Test. Two-way ANOVA was

carried out to test for significance followed by Fisher’s

protected least significant difference test for multiple

comparisons. Heart rate, flow and LVDP measures were

analysed for differences by ANOVA with repeated mea-

sures, followed when significant by post-Tukey test to

assess individual differences. Differences were considered

significant when P \ 0.05.

Results

In Langendorff experiments, the ischaemic area at risk was

similar in all groups with no significant differences

(Fig. 2). In control hearts that had not been subject to RIC,

the infarct size averaged 53 ± 3 % of the ischaemic area at

risk (AAR) (Fig. 3). The infarct size in heart from rats that

had been subjected to RIC was significantly reduced

(27 ± 3 % AAR, n = 6, P \ 0.05, Fig. 3).

The requirement for signalling via the CXCR4 receptor

was examined by injecting rats with AMD3100 prior to

exposure to RIC. This rendered RIC ineffective (40 ± 4 %

vs. 53 ± 3 %, n = 6, n.s., Fig. 3). Indeed, infarct sizes in

AMD ? RIC hearts were significantly larger than RIC

hearts (40 ± 4 % vs. 27 ± 3 %, n = 6, P \ 0.05) (Fig. 3).

AMD3100 had no effect on its own (45 ± 5 %, n = 6)

(Fig. 3).

A 50 % increase plasma SDF-1a levels was detected in

rats subjected to RIC, (890 ± 70 pg/ml compared to

590 ± 50 pg/ml control; n = 8; P \ 0.01) (Fig. 4).

We further investigated the ability of RIC to protect

heart muscle by examining its ability to improve restora-

tion of contractile function after IR, using a model in which

the papillary muscle is removed from the rat heart,

mounted in an apparatus under superfusion, electrically

stimulated, and the developed force measured using a force

transducer. As expected, contractile strength (developed

force) decreased during 30-min hypoxia, and in control

muscle, gradually recovered during reoxygenation to a

peak of 53 ± 13 % of initial force after 75 min (n = 4)Fig. 2 The area at risk (AAR) as a proportion of the left ventricle was

not significantly different between groups

Fig. 3 Remote ischaemic preconditioning (RIC) applied to the rat

hind limb decreases infarct size in rat hearts that are subsequently

removed and subject to ischaemia and reperfusion on a Langendorff,

perfused heart apparatus. Pre-treatment of rats with AMD3100

(AMD), an inhibitor of the SDF-1a receptor, CXCR4, prevents

cardioprotection by RIC (RIC). a Infarct size as percentage of area at

risk (mean ± SEM, n = 6). *P \ 0.05, ***P \ 0.001. b Representa-

tive images of tetrazolium-stained hearts demonstrating infarcted

tissue (white), live tissue (stained red), and non-ischaemic myocar-

dium (blue)

Page 4 of 10 Basic Res Cardiol (2013) 108:377

123

(Fig. 5). Prior RIC improved recovery to 84 ± 5 %

(n = 6, P \ 0.05 vs. control) (Fig. 5). Again, the beneficial

effect of RIC was blocked by AMD3100 (46 ± 7 %,

n = 6) confirming the requirement for signalling via the

CXCR4 receptor. Further validating the protective role of

SDF-1a in this model, SDF-1a given directly to papillary

muscle prior to hypoxia-reoxygenation demonstrated a

significant increase in developed force following hypoxia

and re-oxygenation (89 ± 9 % vs. 55 ± 9 % control,

n = 4, P \ 0.05), and this was blocked by AMD

(60 ± 11 %) (Fig. 6). Finally we verified the presence of

CXCR4, the SDF-1a receptor, in cardiomyocytes and

myocardium by Western blot analysis (Fig. 7). At 67 kDa,

the size of the CXCR4 protein matched that of previously

identified in Western blotting analysis of rat cardiomyo-

cytes [36].

The dipeptidase DPPIV cleaves full-length SDF-1a(1-

67) to SDF-1a(3-67), rendering it unable to bind and

activate the CXCR4 receptor. Therefore, a decrease in

DPPIV by RIC might be posited to cause an increase in the

plasma levels of active SDF-1a. Unfortunately, no method

exists to quantify specifically active SDF-1a(1-67) in

plasma, as all known antibodies recognize both forms. To

try and obtain an indirect assessment of whether

SDF-1a(1-67) was present, the activity of DPPIV was

measured in plasma samples taken from rats immediately

after RIC, or control rats anesthetized for the same period

of time but without exposure to RIC. The level of DPPIV

activity in the plasma was not altered by RIC treatment

(Fig. 8). As a positive control, a DPPIV inhibitor (sitag-

liptin) was given as a pre-treatment and was shown to

completely inhibit DPPIV activity (Fig. 8), demonstrating

that the assay was sensitive.

Discussion

Since the discovery that remote conditioning can protect

the heart from ischaemia and reperfusion injury, attention

has been focused on the possible mechanism by which the

cardioprotective stimulus is transmitted from the limb to

the heart. Evidence to date suggests the involvement of one

or more humoral factors. As a humoral chemokine that is

induced in response to a hypoxia, which has been

Fig. 4 RIC increased plasma SDF-1a levels in rats. SDF-1a was

measured by ELISA in plasma samples from rats immediately after

RIC or control procedures

Fig. 5 RIC improves

contractile recovery of rat heart

papillary muscle that is isolated

and subject to hypoxia and

reoxygenation in vitro (RIC).

Prior injection of rats with

AMD3100 (AMD) eliminates

protection. The gradual loss of

function in papillary muscle

under normoxic conditions is

shown (n = 6 per group

showing mean and SEM)

Basic Res Cardiol (2013) 108:377 Page 5 of 10

123

demonstrated to activate cardioprotective signalling path-

ways, SDF-1a represents a promising candidate for such a

factor. Supporting this hypothesis, we measured a signifi-

cant and reproducible 50 % increase in plasma SDF-1alevels by ELISA immediately following RIC. AMD3100, a

highly specific inhibitor of CXCR4, the sole receptor for

SDF-1a, prevented induction of RIC in rat hearts, both in a

model of infarction in the isolated perfused heart, and in a

model examining contractile function in isolated papillary

muscles. Direct exposure to SDF-1a was confirmed to

induce cardioprotection in papillary muscle, and this was

blocked by AMD3100. The enzyme DPPIV cleaves and

inactivates SDF-1a. Since the plasma activity of DPPIV

was unaltered by RIC the increase in SDF-1a appears not

to be due to an alteration in its plasma half-life. We sug-

gest, therefore, that SDF-1a released from cells activated

during the application of RIC may contribute to the

mechanism of RIC.

In isolated muscle experiments, an increase in contrac-

tile function was observed in RIC or SDF-1a treated groups

(Figs. 6, 7). This is a different result from the isolated heart

experiments in which there were no significant differences

in contractile function (LVDP) between treatment groups

(Fig. 1). One possible explanation for these different

observations is the use of different models, one using just

the papillary muscle and the other the entire heart. Other

protective modalities such as ischaemic preconditioning

have previously been shown to reduce infarct size in the

Langendorff perfused heart without necessarily improving

cardiac function [33]; the isolated perfused heart model

being recognized as a model in which the major endpoint is

infarct size and not function.

The particular model of RIC we employed, in which the

heart was removed from a remotely conditioned animal and

perfused on a Langendorff apparatus, allowed us to spe-

cifically examine the mechanism of the ‘‘induction’’ phase

of RIC in the myocardium, while excluding any possible

direct effect that RIC or SDF-1a might have at reperfusion.

Numerous studies have investigated the potential for SDF-

1a as a treatment post-MI, particularly as a means of

increasing long-term, stem cell migration and homing to

the heart [7, 34, 35, 42, 47]. More recently, it has been

demonstrated that SDF-1a can also protect the heart

acutely without the involvement of stem cells [21, 22],

either after direct administration into the LV cavity in vivo

before ischaemia, or after perfusion through the isolated

heart immediately before ischaemia. As yet there is no

clear consensus as to whether SDF-1a mediates cardio-

protection by activating the MAPK/Erk [21], PI3K/Akt

[21] and/or JAK/STAT3 [22] kinase pathways. However,

these three signalling pathways are well recognized in the

field of cardioprotection as central members of the so-

called ‘‘RISK’’ and ‘‘SAFE’’ pathways, respectively, which

are required for the majority of preconditioning modalities

[14, 32] including RIC [41]. We believe SDF-1a is likely

to be relevant to the human situation since the amino acid

sequence is 100 % conserved with rodents, and it appears

to activate the same signalling pathways [12, 40, 44].

Crucially, SDF-1a activates the same kinases that have

been shown to be involved in RIC. Since some experiments

suggest that RIPC in patients undergoing coronary artery

bypass graft surgery is effective in the presence of isoflu-

orane [29, 30] but not propofol [30], it will be important

to determine whether propofol interferes with the

Fig. 6 SDF-1a prior to

ischaemia improves contractile

recovery of rat heart papillary

muscle that is isolated and

subject to hypoxia and

reoxygenation in vitro (SDF-

1a). Prior injection of rats with

AMD3100 (AMD) eliminates

protection. The gradual loss of

function in papillary muscle

under normoxic conditions is

shown (n = 6 per group

showing mean and SEM)

Page 6 of 10 Basic Res Cardiol (2013) 108:377

123

SDF-1a/CXCR4 signalling axis. This may be relevant for

potential human application of SDF-1a during surgery.

Similarly, release of the humoral factor in RIC appears to

be dependent on preserved neural pathways in diabetic

patients, which, by extension, might suggest that SDF-1arelease is defective in the setting of diabetes [23]. It will be

interesting to investigate these possibilities. These cardio-

protective pathways converge on the mitochondria and

protect the heart by delaying opening of the mitochondrial

permeability transition pore (mPTP), which otherwise

leads to rapid mitochondrial uncoupling, ATP depletion

and cell death [9, 32]. Future work will address the

importance of each of these individual kinase pathways in

the mechanism of RIC via SDF.

The first study to suggest that cardioprotection could be

conferred upon a naı̈ve heart via a protein transferred from a

separate organ which had undergone IPC was performed in

2000 [11]. Proteomic studies subsequently indicated that the

factor released into the blood from a preconditioned organ

is a protein greater than 3.5 kDa [37] and less than 15 kDa

[38] in size. Thus SDF-1a, at 10 kDa, falls into the

expected size range. It also satisfies the requirement for

being able to transfer protection between species [38], as

the protein sequence is 100 % identical between mouse,

rat and human. We observed a 50 % increase in plasma

SDF-1a immediately following the RIC procedure in rats.

Interestingly, remote postconditioning induced with 4

cycles of 5-min occlusion and reperfusion of the abdominal

aorta has been shown to increase SDF-1a levels by 1 h in

female Sprague–Dawley (SD) rats, though this had returned

to baseline by 3 h [24]. A similar procedure in mice resulted

in elevated SDF-1a at 1 h and 3 h [26]. We observed an

increase in plasma SDF-1a concentration of *50 %. Other

studies have shown that plasma SDF-1a levels can be ele-

vated by *100 % [24] to 400 % [26] by ‘‘physiological’’

ischaemia (i.e. preconditioning) although curiously, a

decrease in serum SDF-1a was measured in mice 48 h after

coronary artery ligation [1]. A limitation of our results is

that, in order to confirm that this signalling pathway is

cardioprotective, we used an 80-fold higher concentration

of SDF-1a in vitro than the concentration we measured

in vivo. This was undertaken as we speculated that this

concentration would be necessary to demonstrate protection

in a non-perfused model such as the rat papillary muscle.

This concentration has been used previously to demonstrate

cardioprotection in isolated cardiomyocytes [21]. Further

experiments will be required to confirm that the increase in

plasma SDF-1a concentration that we measured in vivo is

sufficient to induce RIC. Further work will also be required

to determine the precise source of SDF-1a, and target cell

expressing CXCR4. We demonstrated the presence of

CXCR4 within the heart. The presence of more SDF-1a in

the myocardium than in cardiomyocytes suggests that

CXCR4 is expressed in other cell types within the heart in

addition to cardiomyocytes. We hypothesize that the SDF-

1a is released from the ischaemic limb during RIC, travels

to the heart where it binds the CXCR4 receptor and acti-

vates the RISK and/or SAFE pathways of cardioprotection

to delay mPTP opening.

Although AMD3100 significantly reduced cardiopro-

tection by RIC in our model, protection was not completely

eliminated. Since the mechanism of IPC is known to

involve multiple redundant signalling molecules including

Fig. 7 Western blot demonstrating the presence of CXCR4, the

receptor for SDF-1a, in primary adult rat cardiomyocytes (Rat CM)

and in whole rat hearts. Quantification relative to a-tubulin (n = 3,

mean ± SEM, P \ 0.05)

Fig. 8 Plasma DPPIV activity is unchanged in rats after RIC.

Specificity of the assay is demonstrated by the absence of activity in

samples from rats that had been injected with an inhibitor of DPPIV

(sitagliptin) (n = 8 (con and RIC) or 4 (sitagliptin), mean ± SEM,

P \ 0.001 sitagliptin vs. con)

Basic Res Cardiol (2013) 108:377 Page 7 of 10

123

adenosine, bradykinin and opioids [45], it is quite possible

that signalling molecules other than SDF-1a are also

capable of conferring RIC in certain situations. Indeed,

experiments suggest a requirement for IL-10 in delayed

RIC (i.e. after 24 h) in a mouse model [5]. A further lim-

itation of the study is that AMD3100 may have off-target

effects, and has been shown to influence other chemokine

receptors [25].

Since SDF-1a has a plasma half-life of *25 min [31],

we explored the possibility that the increase in SDF-1aplasma levels was due to a decrease in its breakdown. The

majority of SDF-1a is cleaved by the dipeptidase DPPIV,

and consequently, a decrease in DPPIV activity increases

the half-life of SDF-1a [46]. However, we found that

plasma DPPIV activity was unchanged in rats subjected to

RIC. It may be interesting in future to determine whether

pharmacological inhibition of DPPIV can further enhance

the efficacy of RIC. Expression of SDF-1a in vivo

increases in direct proportion to reduced oxygen tension, as

a consequence of the activation of the transcription factor

hypoxia-inducible factor-1 (HIF-1) in hypoxic endothelial

cells [6]. IPC increases levels of SDF-1a mRNA in the

mouse heart [21], and a recent study observed an increase

in HIF1a levels after RIC in patients [2]. As such it is

possible that HIF-1 activation initiates the formation of

SDF-1a during the RIC protocol.

In summary, RIC applied to the limb has been shown to

increase the levels of SDF-1a in the blood. We propose that

this small 10-kDa peptide represents part of the cardio-

protective signal which binds to CXCR4 receptors on the

heart and activates cardioprotective signalling pathways

such as the ‘‘RISK’’ and ‘‘SAFE’’ pathways. With the

identification of a candidate molecule released by RIC, it

may now be possible to develop assays to determine

optimized RIC procedures for clinical use.

Acknowledgments This work was funded by the Rosetrees Trust

and the British Heart Foundation [RG/08/015/26411]. This work was

undertaken at UCLH/UCL who received a proportion of funding from

the Department of Health’s NIHR Biomedical Research Centres

funding scheme of which DM Yellon is a senior investigator.

Conflict of interest None.

References

1. Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ

(2004) Stromal cell-derived factor-1alpha plays a critical role in

stem cell recruitment to the heart after myocardial infarction but

is not sufficient to induce homing in the absence of injury.

Circulation 110:3300–3305. doi:10.1161/01.CIR.0000147780.

30124.CF

2. Albrecht M, Zitta K, Bein B, Wennemuth G, Broch O, Renner J,

Schuett T, Lauer F, Maahs D, Hummitzsch L, Cremer J,

Zacharowski K, Meybohm P (2013) Remote ischemic precondi-

tioning regulates HIF-1alpha levels, apoptosis and inflammation in

heart tissue of cardiosurgical patients: a pilot experimental study.

Basic Res Cardiol 108:314. doi:10.1007/s00395-012-0314-0

3. Botker HE, Kharbanda R, Schmidt MR, Bottcher M, Kaltoft AK,

Terkelsen CJ, Munk K, Andersen NH, Hansen TM, Trautner S,

Lassen JF, Christiansen EH, Krusell LR, Kristensen SD, Thuesen

L, Nielsen SS, Rehling M, Sorensen HT, Redington AN, Nielsen

TT (2010) Remote ischaemic conditioning before hospital

admission, as a complement to angioplasty, and effect on myo-

cardial salvage in patients with acute myocardial infarction: a

randomised trial. Lancet 375:727–734. doi:10.1016/S0140-

6736(09)62001-8

4. Breivik L, Helgeland E, Aarnes EK, Mrdalj J, Jonassen AK

(2011) Remote postconditioning by humoral factors in effluent

from ischemic preconditioned rat hearts is mediated via PI3K/

Akt-dependent cell-survival signaling at reperfusion. Basic Res

Cardiol 106:135–145. doi:10.1007/s00395-010-0133-0

5. Cai ZP, Parajuli N, Zheng X, Becker L (2012) Remote ischemic

preconditioning confers late protection against myocardial

ischemia–reperfusion injury in mice by upregulating interleukin-

10. Basic Res Cardiol 107:277. doi:10.1007/s00395-012-0277-1

6. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas

N, Kleinman ME, Capla JM, Galiano RD, Levine JP, Gurtner GC

(2004) Progenitor cell trafficking is regulated by hypoxic gradi-

ents through HIF-1 induction of SDF-1. Nat Med 10:858–864.

doi:10.1038/nm1075

7. Cheng Z, Ou L, Zhou X, Li F, Jia X, Zhang Y, Liu X, Li Y, Ward

CA, Melo LG, Kong D (2008) Targeted migration of mesen-

chymal stem cells modified with CXCR4 gene to infarcted

myocardium improves cardiac performance. Mol Ther 16:

571–579. doi:10.1038/sj.mt.6300374

8. Cheung MM, Kharbanda RK, Konstantinov IE, Shimizu M,

Frndova H, Li J, Holtby HM, Cox PN, Smallhorn JF, Van Arsdell

GS, Redington AN (2006) Randomized controlled trial of the

effects of remote ischemic preconditioning on children under-

going cardiac surgery: first clinical application in humans. J Am

Coll Cardiol 47:2277–2282. doi:10.1016/j.jacc.2006.01.066

9. Davidson SM, Hausenloy D, Duchen MR, Yellon DM (2006)

Signalling via the reperfusion injury signalling kinase (RISK)

pathway links closure of the mitochondrial permeability transi-

tion pore to cardioprotection. Int J Biochem Cell Biol

38:414–419. doi:10.1016/j.biocel.2005.09.017

10. De Clercq E (2003) The bicyclam AMD3100 story. Nat Rev

Drug Discov 2:581–587. doi:10.1038/nrd1134

11. Dickson EW, Porcaro WA, Fenton RA, Heard SO, Reindhardt

CP, Renzi FP, Przyklenk K (2000) ‘‘Preconditioning at a dis-

tance’’ in the isolated rabbit heart. Acad Emerg Med 7:311–317.

doi:10.1111/j.1553-2712.2000.tb02228.x

12. Ghadge SK, Muhlstedt S, Ozcelik C, Bader M (2011) SDF-1alpha

as a therapeutic stem cell homing factor in myocardial infarction.

Pharmacol Ther 129:97–108. doi:10.1016/j.pharmthera.2010.09.

011

13. Hatse S, Princen K, Bridger G, De Clercq E, Schols D (2002)

Chemokine receptor inhibition by AMD3100 is strictly confined

to CXCR4. FEBS Lett 527:255–262. doi:10.1016/S0014-5793

(02)03143-5

14. Hausenloy DJ, Lecour S, Yellon DM (2011) Reperfusion injury

salvage kinase and survivor activating factor enhancement pro-

survival signaling pathways in ischemic postconditioning: two

sides of the same coin. Antioxid Redox Signal 14:893–907.

doi:10.1089/ars.2010.3360

15. Hausenloy DJ, Mwamure PK, Venugopal V, Harris J, Barnard M,

Grundy E, Ashley E, Vichare S, Di SC, Kolvekar S, Hayward M,

Keogh B, MacAllister RJ, Yellon DM (2007) Effect of remote

ischaemic preconditioning on myocardial injury in patients

Page 8 of 10 Basic Res Cardiol (2013) 108:377

123

undergoing coronary artery bypass graft surgery: a randomised

controlled trial. Lancet 370:575–579. doi:10.1016/S0140-6736

(07)61296-3

16. Hausenloy DJ, Yellon DM (2008) Remote ischaemic precondi-

tioning: underlying mechanisms and clinical application. Car-

diovasc Res 79:377–386. doi:10.1093/cvr/cvn114

17. Hausenloy DJ, Yellon DM (2011) The therapeutic potential of

ischemic conditioning: an update. Nat Rev Cardiol 8:619–629.

doi:10.1038/nrcardio.2011.85

18. Heusch G (2013) Cardioprotection: chances and challenges of its

translation to the clinic. Lancet 381:166–175. doi:10.1016/S0140-

6736(12)60916-7

19. Heusch G, Musiolik J, Kottenberg E, Peters J, Jakob H, Thiel-

mann M (2012) STAT5 activation and cardioprotection by

remote ischemic preconditioning in humans: short communica-

tion. Circ Res 110:111–115. doi:10.1161/CIRCRESAHA.111.

259556

20. Hoole SP, Heck PM, Sharples L, Khan SN, Duehmke R, Densem

CG, Clarke SC, Shapiro LM, Schofield PM, O’Sullivan M, Dutka

DP (2009) Cardiac Remote Ischemic Preconditioning in Coro-

nary Stenting (CRISP Stent) Study: a prospective, randomized

control trial. Circulation 119:820–827. doi:10.1161/CIRCULA-

TIONAHA.108.809723

21. Hu X, Dai S, Wu WJ, Tan W, Zhu X, Mu J, Guo Y, Bolli R,

Rokosh G (2007) Stromal cell derived factor-1 alpha confers

protection against myocardial ischemia/reperfusion injury: role of

the cardiac stromal cell derived factor-1 alpha CXCR4 axis.

Circulation 116:654–663. doi:10.1161/circulationaha.106.672451

22. Huang C, Gu H, Zhang W, Manukyan MC, Shou W, Wang M

(2011) SDF-1/CXCR4 mediates acute protection of cardiac

function through myocardial STAT3 signaling following global

ischemia/reperfusion injury. Am J Physiol Heart Circ Physiol

301:H1496–H1505. doi:10.1152/ajpheart.00365.2011

23. Jensen RV, Stottrup NB, Kristiansen SB, Botker HE (2012)

Release of a humoral circulating cardioprotective factor by

remote ischemic preconditioning is dependent on preserved

neural pathways in diabetic patients. Basic Res Cardiol 107:285.

doi:10.1007/s00395-012-0285-1

24. Jiang Q, Song P, Wang E, Li J, Hu S, Zhang H (2013) Remote

ischemic postconditioning enhances cell retention in the myo-

cardium after intravenous administration of bone marrow mes-

enchymal stromal cells. J Mol Cell Cardiol 56:1–7. doi:10.1016/j.

yjmcc.2012.12.016

25. Kalatskaya I, Berchiche YA, Gravel S, Limberg BJ, Rosenbaum

JS, Heveker N (2009) AMD3100 is a CXCR7 ligand with allo-

steric agonist properties. Mol Pharmacol 75:1240–1247. doi:10.

1124/mol.108.053389

26. Kamota T, Li TS, Morikage N, Murakami M, Ohshima M, Kubo

M, Kobayashi T, Mikamo A, Ikeda Y, Matsuzaki M, Hamano K

(2009) Ischemic pre-conditioning enhances the mobilization and

recruitment of bone marrow stem cells to protect against ische-

mia/reperfusion injury in the late phase. J Am Coll Cardiol

53:1814–1822. doi:10.1016/j.jacc.2009.02.015

27. Kanki S, Segers VF, Wu W, Kakkar R, Gannon J, Sys SU,

Sandrasagra A, Lee RT (2011) Stromal cell-derived factor-1

retention and cardioprotection for ischemic myocardium. Circ

Heart Fail 4:509–518. doi:10.1161/CIRCHEARTFAILURE.110.

960302

28. Kharbanda RK, Mortensen UM, White PA, Kristiansen SB,

Schmidt MR, Hoschtitzky JA, Vogel M, Sorensen K, Redington

AN, MacAllister R (2002) Transient limb ischemia induces

remote ischemic preconditioning in vivo. Circulation

106:2881–2883. doi:10.1161/01.CIR.0000043806.51912.9B

29. Kleinbongard P, Thielmann M, Jakob H, Peters J, Heusch G,

Kottenberg E (2013) Nitroglycerin does not interfere with pro-

tection by remote ischemic preconditioning in patients with

surgical coronary revascularization under isoflurane anesthesia.

Cardiovasc Drugs Ther 29(27):359–361. doi:10.1007/s10557-

013-6451-3

30. Kottenberg E, Thielmann M, Bergmann L, Heine T, Jakob H,

Heusch G, Peters J (2012) Protection by remote ischemic pre-

conditioning during coronary artery bypass graft surgery with

isoflurane but not propofol—a clinical trial. Acta Anaesthesiol

Scand 56:30–38. doi:10.1111/j.1399-6576.2011.02585.x

31. Misra P, Lebeche D, Ly H, Schwarzkopf M, Diaz G, Hajjar RJ,

Schecter AD, Frangioni JV (2008) Quantitation of CXCR4

expression in myocardial infarction using 99mTc-labeled SDF-

1alpha. J Nucl Med 49:963–969. doi:10.2967/jnumed.107.

050054

32. Ovize M, Baxter GF, Di Lisa F, Ferdinandy P, Garcia-Dorado D,

Hausenloy DJ, Heusch G, Vinten-Johansen J, Yellon DM, Schulz

R (2010) Postconditioning and protection from reperfusion

injury: where do we stand? Position paper from the Working

Group of Cellular Biology of the Heart of the European Society

of Cardiology. Cardiovasc Res 87:406–423. doi:10.1093/cvr/

cvq129

33. Sandhu R, Diaz RJ, Wilson GJ (1993) Comparison of ischaemic

preconditioning in blood perfused and buffer perfused isolated

heart models. Cardiovasc Res 27:602–607

34. Sasaki T, Fukazawa R, Ogawa S, Kanno S, Nitta T, Ochi M,

Shimizu K (2007) Stromal cell-derived factor-1alpha improves

infarcted heart function through angiogenesis in mice. Pediatr Int

49:966–971. doi:10.1111/j.1442-200X.2007.02491.x

35. Saxena A, Fish JE, White MD, Yu S, Smyth JW, Shaw RM,

DiMaio JM, Srivastava D (2008) Stromal cell-derived factor-

1alpha is cardioprotective after myocardial infarction. Circulation

117:2224–2231. doi:10.1161/circulationaha.107.694992

36. Segret A, Rucker-Martin C, Pavoine C, Flavigny J, Deroubaix E,

Chatel MA, Lombet A, Renaud JF (2007) Structural localization

and expression of CXCL12 and CXCR4 in rat heart and isolated

cardiac myocytes. J Histochem Cytochem 55:141–150. doi:10.

1369/jhc.6A7050.2006

37. Serejo FC, Rodrigues LF Jr, da Silva Tavares KC, de Carvalho

AC, Nascimento JH (2007) Cardioprotective properties of

humoral factors released from rat hearts subject to ischemic

preconditioning. J Cardiovasc Pharmacol 49:214–220. doi:10.

1097/FJC.0b013e3180325ad9

38. Shimizu M, Tropak M, Diaz RJ, Suto F, Surendra H, Kuzmin E,

Li J, Gross G, Wilson GJ, Callahan J, Redington AN (2009)

Transient limb ischaemia remotely preconditions through a

humoral mechanism acting directly on the myocardium: evidence

suggesting cross-species protection. Clin Sci 117:191–200.

doi:10.1042/CS20080523

39. Smith CC, Lim SY, Wynne AM, Sivaraman V, Davidson SM,

Mocanu MM, Hausenloy DJ, Yellon DM (2011) Failure of the

adipocytokine, resistin, to protect the heart from ischemia–

reperfusion injury. J Cardiovasc Pharmacol Ther 16:63–71.

doi:10.1177/1074248410382232

40. Takahashi M (2010) Role of the SDF-1/CXCR4 system in

myocardial infarction. Circ J 74:418–423. doi:10.1253/circj.CJ-

09-1021

41. Tamareille S, Mateus V, Ghaboura N, Jeanneteau J, Croue A,

Henrion D, Furber A, Prunier F (2011) RISK and SAFE signaling

pathway interactions in remote limb ischemic perconditioning in

combination with local ischemic postconditioning. Basic Res

Cardiol 106:1329–1339. doi:10.1007/s00395-011-0210-z

42. Tang YL, Zhu W, Cheng M, Chen L, Zhang J, Sun T, Kishore R,

Phillips MI, Losordo DW, Qin G (2009) Hypoxic preconditioning

enhances the benefit of cardiac progenitor cell therapy for treat-

ment of myocardial infarction by inducing CXCR4 expression.

Circ Res 104:1209–1216. doi:10.1161/CIRCRESAHA.109.

197723

Basic Res Cardiol (2013) 108:377 Page 9 of 10

123

43. Thielmann M, Kottenberg E, Boengler K, Raffelsieper C, Neu-

haeuser M, Peters J, Jakob H, Heusch G (2010) Remote ischemic

preconditioning reduces myocardial injury after coronary artery

bypass surgery with crystalloid cardioplegic arrest. Basic Res

Cardiol 105:657–664. doi:10.1007/s00395-010-0104-5

44. Wong D, Korz W (2008) Translating an antagonist of chemokine

receptor CXCR4: from bench to bedside. Clin Cancer Res

14:7975–7980. doi:10.1158/1078-0432.CCR-07-4846

45. Yellon DM, Hausenloy DJ (2007) Myocardial reperfusion injury.

N Engl J Med 357:1121–1135. doi:10.1056/NEJMra071667

46. Zaruba MM, Franz WM (2010) Role of the SDF-1-CXCR4 axis

in stem cell-based therapies for ischemic cardiomyopathy. Expert

Opin Biol Ther 10:321–335. doi:10.1517/14712590903460286

47. Zaruba MM, Theiss HD, Vallaster M, Mehl U, Brunner S, David

R, Fischer R, Krieg L, Hirsch E, Huber B, Nathan P, Israel L,

Imhof A, Herbach N, Assmann G, Wanke R, Mueller-Hoecker J,

Steinbeck G, Franz WM (2009) Synergy between CD26/DPP-IV

inhibition and G-CSF improves cardiac function after acute

myocardial infarction. Cell Stem Cell 4:313–323. doi:10.1016/j.

stem.2009.02.013

Page 10 of 10 Basic Res Cardiol (2013) 108:377

123