Vol.2, No.8, 927-934 (2010)
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/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.
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.
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
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
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
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
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].
2.1. 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
Copyright © 2010 SciRes. http://www.scirp.org/journal/HEALTH/Openly accessible at
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,
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.
3.1. Postischemic Recovery of Cardiac
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
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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-
Steady state
(n = 39)
(n = 13)
(n = 13)
(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
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.
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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
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
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.
35 70 140 280 560
Cardiac output, % of initial
35 70 140 280 560
Apelin-12, M
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.
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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.
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
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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.
The authors thank Dr. Zh.D. Bespalova for synthesis of apelin-12 and
discussion of the results.
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