Apelin-12 improves metabolic and functional recovery of rat heart after global ischemia

doi:10.4236/health.2010.28137

Paper Menu >>

Journal Menu >>

Vol.2, No.8, 927-934 (2010)

doi:10.4236/health.2010.28137

HEALTH

Apelin-12 improves metabolic and functional recovery of

rat heart after global ischemia

Oleg I. Pisarenko*, Valentin S. Shulzhenko, Yulia A. Pelogeykina, Irina M. Studneva, Denis N.

Khatri

Russian Cardiology Research-and-Production Complex, Moscow, Russia; *Corresponding Author: olpi@cardio.ru

Received 27 March 2010; revised 24 April 2010; accepted 26 April 2010.

ABSTRACT

This work was designed to explore efficacy of

apelin-12 (A-12) as a cardioprotective agent

when given before ischemia or at reperfusion

using the isolated working heart model. Hearts

of male Wistar rats were subjected to 30-min

stabilization period followed by 35-min global

ischemia and 30-min reperfusion. A short-term

infusion of Krebs-Henseleit buffer (KHB) con-

taining A-12 (35, 70, 140, 280 or 560 M) was ap-

plied prior to ischemia (A-12-I) or at onset of

reperfusion (A-12-R). KHB infusion was used as

control. A-12 infusions induced a dose-depen-

dent increase in recovery of coronary flow,

contractile and pump function during reperfu-

sion, with the largest augmentation of these

indices in the A-12-I group. Both A-12 groups

exhibited a significant reduction of LV diastolic

pressure rise during reperfusion compared with

control. Enhanced functional recovery in the

A-12-I group was combined with a decrease in

LDH leakage in perfusate on early reperfusion

(by 36% vs. control, p < 0.05). Preischemic infu-

sion of 140 M A-12 markedly increased myo-

cardial ATP content, enhanced preservation of

the total adenine nucleotide pool and improved

recovery of the energy charge in reperfused

hearts. There was a trend towards increase in

myocardial phosphocreatine by the end of re-

perfusion in the A-12-I group; however this

benefit did not reach statistical significance. At

the end of reperfusion, myocardial lactate and

lactate/pyruvate ratio were on average 5-fold

lower in A-12-I treated hearts compared with

control ones and did not differ significantly from

the initial values. Therefore, improved cardiac

dysfunction after I/R injury and less cell mem-

brane damage induced by A-12 are associated

with maintaining high energy phosphates, par-

ticularly ATP, in reperfused myocardium. Changes

in energy metabolism may play a role in me-

chanisms of cardioprotection afforded by A-12

during I/R stress.

Keywords: Apelin-12, Rat Heart; Ischemia/

Reperfusion Injury; Energy Metabolism; Cell

Membrane Damage

1. INTRODUCTION

Myocardial ischemia and subsequent reperfusion lead to

formation of a number of intrinsic factors which mediate

the cellular mechanisms of adaptation to altered oxygen

and energy supply [1-3]. One of them is adipocytokine

apelin, recently isolated from bovine stomach extracts

and identified as the endogenous ligand of the human

orphan G protein-coupled receptor APJ. Apelin is a 36-

amino acid peptide derived from 77-amino acid precur-

sor proapelin, for which cDNAs have been cloned from

humans, cattle, rats, and mice [4-6]. Apelin and its re-

ceptor are widely expressed in mammalian tissues; in the

cardiovascular apelin/APJ system was found in the en-

dothelial cells of small intramyocardial and pulmonary

vessels, in coronary arteries, in endocardial endothelium

cells, and in vascular smooth muscle cells [5,7]. Activa-

tion of apelin/APJ receptor system triggers cell-signaling

mechanisms and induces positive inotropic and hypoten-

sive effects in normal and failing myocardium [7-9].

These effects imply an important role of apelin in the

regulation of cardiovascular homeostasis, making it an

attractive target for heart failure therapy.

Only a few studies showed that at least two isoforms

of apelin, apelin-13 and the physiologically less potent

peptide, apelin-36, are capable to attenuate myocardial

ischemia/reperfusion (I/R) injury. Thus, administration

of either of these peptides reduced infarct size and im-

proved contractile function recovery of rat and mouse

hearts after regional or global ischemia [10-12]. In cul-

tured neonatal cardiomyocytes, apelin-13 decreased for-

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

928

mation of reactive oxygen species (ROS) and malonic

dialdehyde (MDA) with simultaneous augmentation of

superoxide dismutase (SOD) activity and reduction of

myocardial LDH leakage under hypoxia/reoxygenation

treatment [12]. Some authors suggest that vasodilator

and antioxidant features of apelin are related to an in-

crease in NO formation due to promoted eNOS expres-

sion [13]. However, а failure of apelin-13 to influence

eNOS phosphorylation was observed in mouse heart

subjected to global ischemia and reperfusion [10]. Ape-

lin-induced suppression of apoptosis, the delayed open-

ing of the mitochondrial permeability transition pore

(mPTP) and reduced cardiomyocyte contracture have

been demonstrated in various experimental myocardial

I/R models, including isolated cardiomyocytes, perfused

hearts and hearts in situ [10,12]. One of the possible

mechanisms whereby the apelin-APJ system may protect

the myocardium form ischemia/reperfusion injury may

be its actions on the reperfusion injury salvage kinase

(RISK) pathway [14,15]. Abolishment of the protective

effects of apelin by PI3K-Akt and ERK specific inhibi-

tors suggests the activation of both kinase cascades by

apelin [10]. These data, and especially inhibition of

mPTP opening [16], indicate an implication of metabolic

component in mechanisms of apelin action on ischemic

heart.

To date, there is no information available regarding

direct effects of the apelin/APJ-system on cardiac energy

metabolism. Therefore, the objective of the present study

was to evaluate alterations in metabolic state of post-

ischemic heart occurring after apelin administration be-

fore ischemia or at onset of reperfusion. To test the po-

tential metabolic importance of apelin, we used a 12

amino acid peptide, apelin-12, which is identical be-

tween human and rats and presumably has higher activ-

ity than longer C-terminal fragments [17].

2. MATERIALS AND METHODS

2.1. Synthesis of Apelin-12

A-12 (chemical structure H-Arg-Pro-Arg-Leu-Ser-His-

Lys-Gly-Pro-Met-Pro-Phe-OH, Mw 1422.7) was synthe-

sized by the automatic solid phase method using an Ap-

plied BioSystems 431A peptide synthesizer (Germany)

and Fmoc technology. The synthesized peptide was puri-

fied by preparative HPLC and identified by 1H-NMR

spectroscopy and mass spectrometry.

2.2. Heart Perfusion

The present investigation conforms to the Guide for Care

and Use of Laboratory Animals published by the US

National Institute of Health (NIH publication No. 85-23;

revised 1985). Male Wistar rats (280-300 g) were

heparinized (500 U i.p.) and anaesthetized with urethane

(1.3 g/kg body weight). Hearts (1.4-1.5 g wet wt) were

excised and immediately placed into ice-cold Krebs-

Henseleit bicarbonate buffer (KHB) until contraction

stopped. The aorta was then cannulated and Langendorff

perfusion was performed at a constant pressure equiva-

lent to 75 cm H2O for 15 min. Working perfusion was

performed according to a modified method of Neely under

constant left atrium pressure and aortic pressure (AP) of

20 and 100 cm H2O, respectively. KHB containing (in

mM): NaCl 118; KCl 4.7; CaCl2 3.0; Na2EDTA 0.5;

KH2PO4 1.2; MgSO4 1.2; NaHCO3 25.0; glucose 11.0.

was oxygenated with a mixture of 95% O2 and 5% CO2;

pH was 7.4 ± 0.1 at 37˚C; it was passed through a 5 m

Millipore filter (Bedford, MA, USA) before use. A nee-

dle was inserted into the left ventricular cavity to regis-

tered LV pressure via a Gould Statham P50 transducer,

SP 1405 monitor and a Gould Brush SP 2010 recorder

(Gould, Oxnard, Ca, USA). The contractile function

index was calculated as the LV developed pressure-heart

rate product (LVDPxHR), where LVDP is the difference

between LV systolic and LV end-diastolic pressure. Car-

diac pump function was assessed by minute volume and

aortic output; stroke was calculated as cardiac output/

HR. Coronary resistance was calculated as AP/coronary

flow [18].

2.3. Experimental Protocol

After preliminary Langendorff perfusion, the hearts were

perfused in working mode for 20 min; the steady state

values of cardiac function were recorded at the end of

this period. Then, the hearts were randomly assigned

onto one of three groups: 1) control (n = 13); 2) A-12

treated before global ischemia (A-12-I, n = 13); 3) A-12

treated at onset of reperfusion (A-12-R, n = 13). After

the steady state period, the control hearts were perfused

in Langendorff mode for 5 min at a constant flow rate of

4 ml/min and then they were subjected to 35-min nor-

mothermic global ischemia followed by 5-min Langen-

dorff perfusion with subsequent 30-min working reper-

fusion. In the A-12-I group, 5-min Langendorff perfu-

sion at a constant flow rate of 4 ml/min with KHB con-

taining A-12 (35, 70, 140, 280 or 560 M) was applied

prior to global ischemia. The hearts of the A-12-R group

were perfused with KHB containing A-12 in the same

mode after global ischemia. After preliminary working

perfusion (steady state) and at the end of reperfusion, the

hearts were freeze-clamped in liquid nitrogen for me-

tabolite analysis. The myocardial effluent was collected

in ice-cold containers during both periods of Langen-

dorff perfusion for immediate measurement of LDH

activity. The total number of hearts used for functional

and metabolic determinations was 49.

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

929

2.4. Tissue Sampling, Metabolite Analysis

and Assay of LDH Activity.

Frozen tissue was quickly homogenized in cooled 6%

perchloric acid (10 ml/g) using an Ultra-Turrax T-25

homogenizer (IKA-Labortechnik, Staufen, Germany), and

the homogenates were centrifuged at 2500 g for 10 min

at 4ºC. The supernatants were then neutralized with 5 M

K2CO3 to pH 7.4 and the extracts were centrifuged after

cooling to remove KClO4 precipitate. Tissue dry weights

were determined by weighing a portion of the pellets

after extraction with perchloric acid and drying over-

night at 110ºC. Concentrations of ATP, ADP, AMP,

phosphocreatine and lactate in neutralized tissue extracts

were determined specrtophotometrically by enzymatic

methods [19-22]. A modified UV-spectroscopy method

was used to assay tissue extracts for pyruvate [23]. De-

termination of metabolite in each tissue extract was per-

formed three times to calculate the average metabolite

concentration in a sample. LDH activity in the myocar-

dial effluent was measured according to the method of

Bergmeyer and Bernt [24] using pyruvate as substrate.

Determination of LDH activity in the perfusate was re-

peated twice in each sample. Enzymes and chemicals

were purchased from Sigma Chemical Co. (St Louis,

MO USA). Solutions were prepared using deionized

water (Milli Ro-4; Milli-Q, Millipore Corp. Bedford,

MA, USA).

2.5. Statistical Analysis

Statistical differences between more than two groups

were evaluated by one-way analysis of variance (AN-

OVA) and followed by Scheffe F-test. Comparisons be-

tween two groups involved use of the Student’s t test. A

p < 0.05 was considered statistically significant.

3. RESULTS

3.1. Postischemic Recovery of Cardiac

Function

Administration of A-12 before global ischemia or at on-

set of reperfusion enhanced recovery of cardiac pump

function during reperfusion. As an example, a dose-

dependent increase in recovery of cardiac output (CO)

by the end of reperfusion is shown on Figure 1(a). The

significant increase in CO recovery was observed after

pre- or postischemic infusion of 70 M A-12 as com-

pared with the control. The differences between CO re-

covery in the experimental groups and control became

more pronounced with an increase in A-12 concentration

in KHB. The maximal response to A-12 was observed at

the concentration of 280 M; at higher concentrations a

dose-effect curve reached a plateau. Within the range of

70-560 M, the peptide administration prior to ischemia

was more effective than after it. Thus, the maximal res-

toration of CO after infusion 560 M A-12 were 86 ±

9% and 59 ± 6% of the steady state value in the group

A-12-I and A-12-R, correspondingly, in comparison with

32 ± 2% in the control (P < 0.02-0.01). А similar dose-

dependent effects were obtained for recovery of the

LVDPxHR product during reperfusion in both experimen-

tal groups (Figure 1(b)).

Recovery of cardiac and coronary function indices at

the end of reperfusion after administration of 140 M A

-12 before and after of ischemia is presented in Table 1.

In addition to CO, aortic output and stroke volume were

also significantly higher in the A-12-I group than in the

A-12-R group and in control. The especially greater dif-

ferences between the groups were noted for aortic output:

in the A-12-I group its recovery was twice more effec-

tive than in the A-12-R group and 30 times higher than

in control. An enhanced recovery of cardiac pump func-

tion in A-12 treated hearts was accompanied by an aug-

mented restoration of the LVDPxHR product. This effect

was due to higher recovery of HR and in both A-12

groups comparing with control. A significant increase in

the values of LVDP was in turn caused by a marked re-

duction of LV diastolic pressure during reperfusion

(Figure 2). Beneficial effects of A-12 on cardiac func-

tion were accompanied by the peptide influence on the

coronary system. Thus, both of A-12 groups exhibited a

significant increase in coronary flow with concomitant

reduction in coronary resistance in comparison with

control (Table 1) indicating a participation of coronary

vessels in response to A-12 administration.

3.2. The Energy State of Reperfused Hearts

Changes in the myocardial content of adenine nucleo-

tides and the end products of anaerobic glycolysis, lac-

tate and pyruvate, at the end of reperfusion caused by

preischemic infusion of 140 M A-12 are compared with

the initial myocardial levels of these metabolites in Ta-

ble 2. By the end of reperfusion, the control group

showed a profound decrease in the ATP content (to 37 ±

7% of the initial value) with a simultaneous increase in

ADP and AMP levels (on the average 1.7 and 5.6 times,

respectively). These changes indicated the preferred

degradation of adenine nucleotides, whose total myocar-

dial pool (AN = ATP + ADP + AMP) was lowered to

58 ± 3% of the steady state value. The lactate and pyru-

vate contents were, correspondingly, 5- and 1.3-fold

higher than the normal values, thus indicating an inhibi-

tion of glucose oxidation during reperfusion. Admini-

stration of 140 M A-12 before ischemia enhanced ATP

preservation by 60 ± 3% and twice decreased AMP con-

tent in the reperfused hearts. As a result the myocardial

AN pool was preserved considerably better than in the

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

930

control and composed 81 ± 6% of the steady state value.

Redistribution in the adenine nucleotide content consid-

erably increased the total energy charge (EC = (ATP +

0.5ADP)/ATP + ADP + AMP) in the A-12-I group as

compared with the control. The observed improvement

in the energy state of reperfused hearts was combined

with а decrease in myocardial lactate content to the

steady state value (Table 2). Although the myocardial

pyruvate level in the A-12-I group remained raised at the

end of reperfusion, the lactate/pyruvate ratio was 5 times

lower than in the control and did not differ significantly

from the steady state value.

Preischemic administration of 140 M A-12 did not

significantly enhance recovery of phosphocreatine (PCr):

by the end of reperfusion its myocardial content was

14.95 ± 1.32 mol/g of dry wt vs. 12.02 ± 2.61 mol/g

of dry wt in the control. The total creatine pool (Cr =

PCr + creatine) in A-12-treated hearts did not differ sig-

Table 1. Effects of apelin-12 administration before or after global ischemia on functional recovery of isolated rat at the end of reper-

fusion.

Steady state

(n = 39)

Control

(n = 13)

A-12-R

(n = 13)

А-12-I

(n = 13)

Coronary flow,

ml/min 19.1 ± 2.0 14.5 ± 0.4 15.9 ± 1.1 17.8 ± 0.9 a

Perfusion pressure,

mm Hg 64 ± 6 60 ± 1 61 ± 1 63 ± 1 a

Coronary resistance,

mm Hg/ml 3.46 ± 0.04 4.19 ± 0.07 3.98 ± 0.21 3.78 ± 0.17 a

LV systolic pressure,

mm Hg 102 ± 1 70 ± 1 80 ± 3 a 92 ± 3 a b

LV diastolic pressure,

mm Hg -2 ± 1 8 ± 1 5 ± 1 a 1 ± 1 a b

LV developed pressure,

mm Hg 106 ± 1 62 ± 1 75 ± 4 a 92 ± 4 a b

Heart rate,

beat/min 310 ± 2 242 ± 3 264 ± 6 a 288 ± 6 a b

LVDP x HR,

mm Hg/min 33062 ± 624 14877 ± 561 20167 ± 1653 a 26780 ± 1653 a b

Aortic output,

ml/min 28.1 ± 3.0 0.6 ± 0.3 8.7 ± 0.8 a 19.1 ± 1.3 a b

Cardiac output,

ml 46.2 ± 1.1 14.8 ± 0.9 24.0 ± 2.3 a 35.5 ± 2.2 a b

Stroke volume, l 149 ± 1 61 + 3 91 + 7 a 127 + 4 a b

The hearts were perfused as indicated in Materials and methods. Steady state, 20-min preliminary working perfusion; Control, 5-min Langendorff

perfusion + 35-min global ischemia + 5-min Langendorff perfusion + 30-min working reperfusion; A-12-R, 5-min Langendorff perfusion with 140

M A-12 after global ischemia; A-12-I, 5-min Langendorff perfusion with 140 M A-12 before global ischemia. Data are the mean ± SEM. a p < 0.05

vs. control group; b p < 0.05 vs. A-12-R group.

Table 2 Effects of 140 M apelin-12 infusion before global ischemia on myocardial content of metabolites and indices of myocar-

dial energy state at the end of reperfusion.

Steady state Control А-12-I

АTP 22.42 ± 2.06 8.30 ± 1.73 а 13.11 ±0.69 а b

АDP 2.80 ± 0.12 4.86 ± 0.28 а 5.90 ± 0.28 а

АMP 0.71 ± 0.01 3.97 ± 0.74а 1.92 ± 0.06 а b

АN 25.93 ± 1.45 17.13 ± 1.42 а 20.94 ± 0.92 а b

AT P/ АDP 8.00 + 0.10 1.72 ± 0.35 а 2.23 ±0.07 а

EC 0.91 ± 0.02 0.60 ± 0.06 а 0.77 ± 0.01 а b

Lactate 1.72 ± 0.19 10.48 ± 3.08а 2.12 ± 0.48 b

Pyruvate 0.18 ± 0.02 0.23 ± 0.03 0.22 ± 0.02

Lactate/Pyruvate 9.55 ± 0.98 45.56 ± 7.34 а 9.64 ± 1.25 b

Steady state, 20-min preliminary working perfusion; Control, 5-min Langendorff perfusion + 35-min global ischemia + 5-min Langendorff perfusion

+ 30-min working reperfusion; A-12-I, 5-min Langendorff perfusion with 140 M A-12 before global ischemia. Data are the mean ± SEM for 10

experiments. Metabolite contents are expressed in (mol/g dry wt). a p < 0.05 vs. steady state; b p < 0.05 vs. control. АN = ATP + ADP + AMP. The

energy charge (EC) = (ATP + 0.5ADP)ATP + ADP + AMP.

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

931

Openly accessible at

nificantly from this index in the control and in the steady

state being 59.65 ± 1 56, 56.65 ± 3.89 and 59.26 ± 1.87

mol/g of dry wt, correspondently.

3.3. Lactate Dehydrogenase Leakage

The ability of A-12 to influence cell membrane damage

was assessed by changes in LDH release into the myocar-

dial effluent before and after global ischemia (Table 3).

For the 5-min period of 140 M A-12 infusion prior to

ischemia, LDH leakage did not differ significantly from

that one in control. Therefore A-12 administration did

cause damage to the sarcolemma of nonischemic car-

diomyocytes. During the 5-min period after ischemia,

the release of LDH activity in the perfusate of the con-

trol group was increased on average 2.6 times compared

with this value before ischemia indicating I/R membrane

damage. However in the A-12-I group, the postischemic

LDH leakage was reduced by 40% compared with con-

trol. This finding suggested fewer membrane defects

determining a release of cytoplasmic LDH from the

myocardium.

4. DISCUSSION

The present study demonstrates, for the first time, car-

dioprotective properties of exogenous A-12 in isolated

working rat heart subjected to global ischemia and re-

perfusion. They are manifested by enhanced contractile

and pump function recovery and a better restoration of

coronary flow, which are more effective after the peptide

administration before ischemia than at onset of reperfu-

sion. (Figure 1, Table 1). The оbtained data are in broad

agreement with the previous observations demonstrating

a reduction of ischemia-reperfusion injury induced by

exogenous apelin-13, Pyr1-apelin-13 and apelin-36 in

Langendorff perfused mouse and rat hearts [10-12,25]. It

is essential that a higher functional level of postischemic

hearts protected by A-12 in our experiences is combined

with augmented metabolic state. Probably this beneficial

effect is associated with enhanced oxidation of glucose,

the main energy substrate of isolated perfused hearts.

Indeed, A-12 treated hearts exhibit substantially reduced

lactate/pyruvate ratio as compared with control and the

normal myocardial lactate content at the end of reperfu-

sion. In parallel, A-12 administration exerts an energy

conservation effect evidenced by higher myocardial ATP

and AN contents and enhanced EC of postischemic

cardiomyocytes. In addition, augmentation of energy

state of postischemic hearts by pretreatment with 140

M A-12 is accompanied by less cell membrane damage

on early reperfusion (Тable 3). Taken together, these

findings indicate that alterations in myocardial metabo-

lism induced by apelin may play a role in improving

cardiac dysfunction after ischemia and reperfusion

stress.

Table 3. Effects of 140 M apelin-12 infusion on LDH

leakage before and after global ischemia.

Before ischemia After ischemia

Control (n = 6) 28.60 ± 3.01 73.64 ± 5.06 б

А-12-I (n = 7) 24.28 ± 2.73 53.56 ± 6.52 а b

Control, 5-min Langendorff perfusion + 35-min global ischemia +

5-min Langendorff perfusion + 30-min working reperfusion; A-12-I,

5-min infusion of 140 M A-12 before global ischemia. Values are the

means + SEM and are expressed in IU/g dry wt. for 5-min Langendorff

perfusion before or after global ischemia. a p<0.05 vs. control group;

b p < 0.05 vs. the value before ischemia.

100

35 70 140 280 560

Cardiac output, % of initial

100

35 70 140 280 560

Apelin-12, M

LVDP x HR, % of initial

Figure 1. Effects of A-12 concentration in KHB on recovery

of minute volume (A) and the LVDP-HR product (B) at the

end of reperfusion. 1) 5-min Langendorff perfusion + 35-min

global ischemia + 5-min Langendorff perfusion + 30-min

working reperfusion (control); 2) 5-min Langendorff perfu-

sion with A-12 after global ischemia (A-12-R); 3) 5-min

Langendorff perfusion with A-12 before global ischemia

(A-12-I). Data are the mean + SEM for 12-15 experiments.

* p < 0.05 vs. control; # p < 0.05 vs. A-12-R.

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

932

Openly accessible at

0104050607080

-4

Time, min

LV diastolic pressure, mm Hg

Ischemia Reperfusion

Figure 2. Effects of A-12 administration before or after

global ischemia on recovery of LV diastolic pressure during

reperfusion. 1) 5-min Langendorff perfusion + 35-min

global ischemia + 5-min Langendorff perfusion + 30-min

working reperfusion (control); 2) 5-min Langendorff perfu-

sion with 140 M A-12 after global ischemia (A-12-R); 3)

5-min Langendorff perfusion with 140 M A-12 before

global ischemia (A-12-I). Data are the mean+ SEM for

12-15 experiments. * p < 0.05 vs. control; # p < 0.05 vs.

A-12-R.

The current studies evaluating the mechanisms wh-

ereby apelin confer protection against I/R injury have

yielded mixed results. Wide discrepancy relates to varia-

tions in apelin/APJ expression. In ventricular cardiomyo-

cytes isolated from rats, apelin expression is increased

under hypoxia presumably via hypoxia-inducible fac-

tor-mediated pathway [12,26]. Apelin and APJ are up-

regulated in the heart and skeletal muscle following

myocardial injury in the murine model of LAD occlu-

sion in vivo [27]. Accordingly, endogenous myocardial

apelin and APJ expression are increased in failing rat

hearts in compensation for ischemic cardiomyopathy

[28]. In contrast, apelin content in plasma, atrial and

ventricular myocardium and APJ gene expression de-

crease in myocardial injury induced by repeated isopro-

terenol injections in rats [29]. The endogenous apelin/

APJ system is compensatory up-regulated and ultimately

down-regulated during sustained myocardial ischemia in

vitro which was mimicked by glucose deprivation [30].

In isolated rat heart, apelin and APJ mRNA are up-

regulated during ischemia but return to the control levels

following reperfusion [11]. Based on these conflicting

data we suggest that in acute ischemic myocardial injury,

the endogenous apelin/APJ system may have a protec-

tive role. However the endogenous apelin production is

insufficient for activation of the APJ receptors to reduce

myocardial ischemia-reperfusion injury. This assumption

is consistent with cardioprotective activity of exogenous

isoforms of apelin revealed in different experimental

models [7,10-12,28-30].

Effects of exogenous apelin are mediated partly by ac-

tivation of components of the reperfusion injury salvage

kinase (RISK) pathway, phosphatidylinositol-3-OH kinase

(PI3K)/Akt, p44/42 mitogen-activated protein kinase

(MAPK) extracellular signal-regulated MAPK (ERK1/2)

[10,14,16,31]. This is confirmed by that fact that the

inhibitors of PI3K-Akt and p44/42 phosphorylation,

LY294002 and UO126, respectively, abolished reduction

of infarct size induced by apelin-13 in vitro [10]. Simi-

larly, addition of wortmannin and PD098059, the inhibi-

tors of PI3K/Akt and ERK1/2, to culture medium during

hypoxia/reoxygenation, suppressed cardiomyocyte vi-

ability provided by apelin-13 [12]. Further evidence that

apelin may act via activation of the RISK pathway was

obtained from the findings concerning the mPTP open-

ing, which causes the energy collapse and irreversible

cardiomyocyte damage [16,32]. Thus, while apelin-13

delayed the times until mitochondrial membrane depo-

larization and rigor contracture, LY294002 and mitogen-

activated protein kinase (MEK) inhibitor 1 blocked these

effects [10]. Anti-apoptotic effects of apelin-13 in glu-

cose-deprived cardiomyocytes involve activation of the

PI3K/Akt and the mammalian target of rapamycin

(mTOR) pathways [30]. As follows from the study by

Kleinz and Baxter [11], the RISK cascades are not al-

ways used for myocardial protection afforded by apelin.

These authors showed that apelin-13 administered dur-

ing reperfusion significantly reduced infarct size in rat

hearts subjected to coronary occlusion followed by re-

perfusion. However this protective effect was not abol-

ished by co-administration of the PI3K/Akt inhibitor

wortmannin or the P70S6 kinase inhibitor rapamycin.

Therefore, apelin may exhibit cardioprotection using al-

ternative mediators and signaling pathways.

Elevation of eNOS expression induced by apelin [8,

12] may lead to increase in NO formation, contributing

to recovery of cardiac function and metabolism after

ischemia. In our study, it is indirectly confirmed by sig-

nificant improvement of coronary flow in the A-12-I

group during reperfusion (Table 1). Involvement of the

L-Arg/NOS/NO pathway in vascular function regulation

by apelin was noted earlier Tatemoto et al. [4]. They

demonstrated that in anaesthetized rats the hypotensive

effect of apelin-12 (to a greater extent than apelin-13 and

apelin-36) is accompanied by an increase in the total ni-

trite and nitrate in plasma, and is abrogated in the pres-

ence of N-nitro-L-arginine methyl ester (L-NAME), a

NOS inhibitor. Subsequently, a dose-dependent reduc-

tion of the mean arterial pressure during apelin infusion

was documented in conscious rats [33]. It is equally im-

portant that during myocardial ischemia and reperfusion,

NO generated by vascular and endocardial endothelial

NOS may attenuate oxidative stress by scavenging reac-

tive oxygen species (ROS) and reduce cell death via

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

933

Openly accessible at

inhibition of mPTP [34,35]. The role of NO, formed

under apelin administration, in reducing I/R injury so far

remains unclear. However, the antioxidant properties of

exogenous apelin-13 were confirmed by parallel de-

crease in ROS and MDA formation in the isolated rat

heart and cardiomyocyte culture during simulation of

ischemic and reperfusion stress [12]. It is possible that

these effects were due not only to eNOS expression, but

also to apelin-stimulated increase in SOD activity, which

is inhibited in myocardial tissue by I/R [12,36].

In addition to the above mechanisms the results of our

study clearly indicate that enhanced energy state of

reperfused myocardium is implicated in amelioration of

I/R injury by apelin. In fact, a better preservation of

myocardial adenine nucleotides, and especially ATP,

may be of critical importance for maintaining cell mem-

brane integrity and ion homeostasis, preventing contrac-

ture and ROS generation during reperfusion [37,38].

Presumably a more effective postischemic restoration of

energy metabolism was related to promoted glucose

oxidation in A-12 treated hearts (Table 2). Various stud-

ies point out an emerging role of apelin in glucose me-

tabolism [39]. In particular, it was shown that apelin

stimulates glucose utilization in normal and insulin- re-

sistant mice [40]. In the heart and skeletal muscle, apelin

effects on glucose uptake have been suggested to be as-

sociated with the activation of eNOS, AMP-activated

protein kinase (AMPK) and Akt-dependent pathways

[41,43]. Therefore apelin-stimulated alterations in glu-

cose metabolism may represent a promising approach

for correcting metabolic disorders induced by ischemia

and reperfusion.

In conclusion, the present study revealed the ability of

exogenous apelin-12 to improve cardiac dysfunction, sar-

colemma integrity and myocardial metabolic state after

I/R injury. Although apelin isoforms with shorter C-

terminal fragments than apelin-36 have higher bioactiv-

ity, they may be subjected to enzymatic degradation in

vivo with formation of inactive forms. Synthesis of

modified analogues of apelin is able to increase their

resistance to the action of aminopeptidases and lead to

the development of pharmacological agonists for APJ

receptor. This intriguing possibility may provide new

therapeutic tools for treatment acute coronary syndrome

and heart failure.

5. ACKNOWLEDGEMENTS

The authors thank Dr. Zh.D. Bespalova for synthesis of apelin-12 and

discussion of the results.

REFERENCES

[1] Verma, S., Fedak, P.W., Weisel, R. D., Butany, J., Rao, V.

and Maitland, A. (2002) Fundamentals of reperfusion in-

jury for the clinical cardiologist. Circulation, 105(20),

2332-2336.

[2] Infanger, M., Faramarzi, S., Grosst, J., Kurth, E., Ulbrich,

C. and Bauer J. (2007) Expression of vascular endothe-

lial growth factor and receptor tyrosine kinases in cardiac

ischemia/reperfusion injury. Cardiovascular Pathology,

16(5), 291-299.

[3] Smith. C.C., Mocanu, M.M., Davidson, S. M., Wynne, A.

M., Simpkin, J. C. and Yellon D. M. (2006) Leptin, the

obesity-associated hormone, exhibits direct cardioprotec-

tive effects. British Journal of Pharmacology, 149(1),

5-13.

[4] Tatemoto, K., Hosoya, M., Habata, Y., Fujii, R., Kake-

gawa, T., Zou, M. X., Kawamata, Y., Fukusumi, S.,

Hinuma, S., Kitada, C., Kurokawa, T., Onda, H. and Fu-

jino, M. (1998) Isolation and characterization of a novel

endogenous peptide ligand for the human APJ receptor.

Biochemical and Biophysical Research Communications,

251(2), 471-476.

[5] Boucher, J., Masri, B., Daviaud, D., Gesta S., Guigne, C.

and Mazzucotelli, A. (2005) Apelin, a newly identified

adipokine up-regulated by insulin and obesity. Endocri-

nology, 146, 1764-1771.

[6] Lee, D. K., Cheng, R., Nguyen, T., Fan, T., Kariyawasam,

A. P., Liu, Y., Osmond, D. H., George, S. R. and O’Dowd,

B.F. (2000) Characterization of apelin, the ligand for the

APJ receptor. Journal of Neurochemistry, 74(1), 34-41.

[7] Berry, M.F., Pirolli, T.J., Jayasankar, V., Burdick, J.,

Morine, K.J. and Gardner, T.J. (2004) Apelin has in vivo

inotropic effects on normal and failing hearts. Circula-

tion, 110(Supplement II), II187-II193.

[8] Tatemoto, K., Takayama, K., Zou, M.X., Kumaki, I.,

Zhang, W. and Kumano, K. (2001) The novel peptide

apelin lowers blood pressure via a nitric oxide-dependent

mechanism. Regulatory Peptides, 99(2-3), 87-92.

[9] Chen, M.M., Ashley, E.A., Deng, D.X., Tsalenko, A.,

Deng, A. and Tabibiazar, R. (2003) Novel role for the

potent endogenous inotrope apelin in human cardiac

dysfunction. Circulation, 108(12), 1432-1439.

[10] Simpkin, J. C., Yellon, D. M., Davidson, S.M., Lim, S. Y.,

Wynne, A. M. and Smith, C. C. (2007) Apelin-13 and

apelin-36 exhibit direct cardioprotective activity against

ischemia-reperfusion injury. Basic Research in Cardiol-

ogy, 102(6), 518-528.

[11] Kleinz, M.J. and Baxter, G.F. (2008) Apelin reduces

myocardial reperfusion injury independently of PI3K/Akt

and P70S6 kinase. Regulatory Peptides, 146(1-3), 271-

277.

[12] Zeng, X. J., Zhang, L. K., Wang, H. X., Lu, L. Q., Ma, L.

Q. and Tang, C.S. (2009) Apelin protects heart against

ischemia/reperfusion injury in rat. Peptides, 30(6), 1144-

1152.

[13] Jia, Y. X., Lu, Z.F., Zhang, J., Pan, C. S., Yang, J.H. and

Zhao, J. (2007) Apelin activates L-arginine/nitric oxide

synthase/nitric oxide pathway in rat aortas. Peptides,

28(10), 2023-2029.

[14] Masri, B., Lahlou, H., Mazarguil, H., Knibiehler, B. and

Audigier, Y. (2002) Apelin (65-77) activates extracellular

signal-regulated kinases via a PTX-sensitive G protein.

Biochemical and Biophysical Research Communications,

290(1), 539-545.

O. I. Pisarenko et al. / HEALTH 2 (2010) 927-934

934

[15] Masri, B., Knibiehler, B. and Audigier, Y. (2005) Apelin

signaling: A promising pathway from cloning to phar-

macology. Cell Signaling, 17(4), 415-426.

[16] Tsang, A., Hausenloy, D.J., Mocanu, M.M. and Yellon,

D.M. (2004) Postconditioning: A form of “modified

reperfusion” protects the myocardium by activating the

phosphatidylinositol 3-kinase-Akt pathway. Circulation

Research, 95(3), 230-232.

[17] Kawamata, Y., Habata, Y. and Fukusumi, S. (2001) Mo-

lecular properties of apelin: tissue distribution and re-

ceptor binding. Biochimica et Biophysica Acta, 1538(2-3),

162-171.

[18] Pisarenko, O. I., Shulzhenko, V. S. and Studneva, I.M.

(1999) Metabolic effects of carbachol and ischaemic

preconditioning in isolated rat heart. Clinical and Ex-

perimental Pharmacology and Physiology, 26(1), 26-31.

[19] Lamprecht, W. and Trautschold, I. (1974) Creatine phos-

phate. Determination with CK, HK and G6P-DH. In:

Bergmeyer, H. U., Ed., Methods of Enzymatic Analysis.

Academic Press, New York, 1777-1781.

[20] Jaworek, D., Gruber, W. and Bergmeyer, H.U. (1974)

Adenosine-5’-diphosphate and adenosine-5’-monophos-

phate. In: Bergmeyer, H. U., Ed., Methods of Enzymatic

Analysis. Academic Press, New York, 2127-2131.

[21] Bernt, E., Bergmeyer, H. U. and Mollering H. (1974)

Creatine. In: Bergmeyer, H.U., Ed., Methods of Enzy-

matic Analysis. Academic Press, New York, 1772-1776.

[22] Gutman, I., Wahlenfeld, A.W.L. (1974) L-(+)-Lactate.

Determination with LDH and NAD. In: Bergmeyer, H.U.,

Ed., Methods of Enzymatic Analysis. Academic Press,

New York, 1464-1467.

[23] Bucher, T., Czok, R., Lamprecht, W. and Latzko E. (1963)

Pyruvate. In: Bergmeyer, H.U., Ed., Methods of enzy-

matic analysis. Academic Press, New York, 2253-2259.

[24] Bergmeyer, H.U. and Bernt, E. (1974) Lactate dehydro-

genase. UV-assay with pyruvate and NADH. In: Berg-

meyer, H.U., Ed., Methods of enzymatic analysis. Aca-

demic Press, New York, 574-578.

[25] Zeng, X.J., Wang, H.X., Lu, L.Q., Hao, G., Wang, X.Y.

and Ma, L.Q. (2007) Effects and mechanisms of ape-

lin-13 on ischemia/reperfusion injury in rat heart. Chini-

cal Pharmacology Bull, 23(1), 82-85.

[26] Ronkainen, V.P., Ronkainen, J.J. and Hanninen, S.L.

(2007) Hypoxia inducible factor regulates the cardiac

expression and secretion of apelin. Journal of the Fed-

eration of American Societies for Experimental Biology,

21(8), 1821-1830.

[27] Sheikh, A.Y., Chun, H.J. and Glassford, A.J. (2008) In

vivo genetic profiling and cellular localization of apelin

reveals a hypoxia-sensitive, endothelial centered pathway

activated in ischemic heart failure. American Journal of

Physiological Heart and Circulatory Physiology, 294(1),

H88-H98.

[28] Atluri, P., Morine, K.J. and Liao, G.P. (2007) Ischemic

heart failure enhances endogenous myocardial apelin and

A.P.J. receptor expression. Cellular and Molecular Biol-

ogy Letter, 12(1), 127-138.

[29] Jia, Y.X., Pan, C.S., Zhang, J., Geng, B., Zhao, J. and

Gerns, H. (2006) Apelin protects myocardial injury in-

duced by isoproterenol in rats. Regulatory Peptides,

133(1-3), 147-154.

[30] Zhang, Z., Yu, B. and Tao, G.-Z. (2009) Apelin protects

against cardiomyocyte apoptosis induced by glucose

deprivation. Chiniese Medical Journal, 122(19), 2360-

2365.

[31] Chandrasekaran, B., Dar, O. and McDonagh, T. (2008)

The role of apelin in cardiovascular function and heart

failure. European Journal of Heart Failure, 10(8),

725-732.

[32] Smith, C.C., Mocanu, M.M. and Bowen, J. (2007) Tem-

poral changes in myocardial salvage kinases during

reperfusion following ischemia: Studies involving the

cardioprotective adipocytokine apelin. Cardiovascular

Drugs and Therapy, 21(6), 409-414.

[33] Сheng, X., Cheng, X.S. and Pang, C.C. (2003) Venous

dilator effect of apelin, an endogenous peptide ligand for

the orphan APJ receptor, in conscious rats. European

Journal of Pharmacology, 470(3), 171-175.

[34] Rastaldo, R., Pagliaro, P., Cappello, S., Penna, C., Man-

cardi, D., Westerhof, N. and Losano, G. (2005) Nitric

oxide and cardiac function. Life Sciences, 81(10), 779-

793.

[35] Shultz, R., Kelm, M. and Heusch, G. (2004) Nitric oxide

in myocardial ischemia/reperfusion injury. Cardiovascu-

lar Research, 61(3), 402-413.

[36] Ferrari, R., Ceconi, C., Curello, S., Cargnoni, A., Alfieri,

O., Pardini, A., Marzollo, P. and Visioli, O. (1991) Oxy-

gen free radicals and myocardial damage: protective role

of thiol-containing agents. The American Journal of

Medicine, 91(3), 95S-105S.

[37] Lipasti, J.A., Nevalainen, T.J., Alanen, K.A. and Tol-

vanen, M.A. (1984) Anaerobic glycolysis and the devel-

opment of ischaemic contracture in isolated rat heart.

Cardiovascular Research, 18(3), 145-148.

[38] Pisarenko, O.I., Lakomkin, V.L., Studneva, I.M., Ti-

moshin, A.A., Kuzmin, A.I., Ruuge, E.K. and Kapelko,

V.I. (1994) Allopurinol-enhanced postischemic recovery

in the isolated rat heart involves repletion of high-energy

phosphates. Biochemical Medicine and Metabolic Biol-

ogy, 51(1), 16-26.

[39] Rosen, E.D. and Spiegelman, B.M. (2006) Adipocytes as

regulatord of energy balance and glucose homeostasis.

Nature, 444(7120), 847-853.

[40] Dray, C., Knauf, C., Daviaud, D., Waget, A., Boucher, J.,

Buleon, M., Cani, P.D., Attane, C. and Guigne, C. (2008)

Apelin stimulates glucose utilization in normal and obese

insulin-resistant mice. Cell Metabolism, 8(5), 437-445.

[41] Li, J., Hu, X., Selvakumar, P., Russell, R.R., Cushman,

S.W., Holman, G.D. and Young, L.H. (2004) Role of the

nitric oxide pathway in AMPK-mediated glucose uptake

and GLUT4 translocation in heart muscle. American

Journal of Physiology—Endocrinology Metabolism,

287(5), E834-E841.

[42] Yue, P., Jin, H., Aillaud, M., Deng, A.C., Azuma, J.,

Asagami, T. and Kundu, R. K. (2010) Apelin is necessary

for the maintenance of insulin sensitivity. American

Journal of Physiology—Endocrinology Metabolism,

298(1), E59-E67.