[Pyr1]apelin-13 identified as the predominant apelin isoform in the human heart: vasoactive mechanisms and inotropic action in disease.

Abstract

Apelin receptors, present on vascular smooth muscle cells, endothelium, and cardiomyocytes, are activated by the family of apelin peptides to elicit cardiovascular effects in experimental animals, but functional activity in humans has not been studied in detail. We detected low levels of apelin immunoreactivity in plasma of volunteers consistent with an autocrine/paracrine action and detected apelin immunoreactivity in the supernatant from human cultured endothelial cells. We found that [Pyr(1)]apelin-13 was the predominant isoform in cardiac tissue from patients with coronary artery disease. We tested the hypothesis that apelins have vascular and cardiac actions in human tissues in vitro and compared responses to [Pyr(1)]apelin-13, apelin-13, and apelin-36. In endothelium-intact mammary artery, all 3 of the apelins induced concentration-dependent vasodilatation with comparable potency (EC(50): 0.6 to 1.6 nM; maximum response: 40% to 50%). Vasodilatation was abolished after endothelial removal or preincubation with indomethacin but was unaffected by preincubation with N(G)-nitro-L-arginine methyl ester, indicating involvement of prostanoids but not NO in dilatation by apelins in this patient group. Apelins were potent constrictors of endothelium-denuded saphenous vein (EC(50): 0.6 to 1.6 nM; maximum response: 17% to 26%) and mammary artery ([Pyr(1)]apelin-13; EC(50): 0.2 nM; maximum response: 29%). In paced atrial strips, all 3 of the peptides increased the force of contraction with subnanomolar potencies (EC(50): 40 to 125 pM). For the first time, we demonstrate that the 3 principal forms of apelin have comparable potency and efficacy in human cardiovascular tissues. Apelins are potent endothelium-dependent vasodilators acting via a prostanoid-dependent mechanism; however, removal of the endothelium revealed direct vasoconstrictor actions in both the artery and vein. Furthermore, in human cardiac tissue, the apelin peptides are among the most potent endogenous positive inotropic agents yet reported.

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ISSN: 1524-4563
Copyright © 2009 American Heart Association. All rights reserved. Print ISSN: 0194-911X. Online
72514
Hypertension is published by the American Heart Association. 7272 Greenville Avenue, Dallas, TX
DOI: 10.1161/HYPERTENSIONAHA.109.134619
published online Jul 13, 2009;
Hypertension
Janet J. Maguire, Matthias J. Kleinz, Sarah L. Pitkin and Anthony P. Davenport

Heart. Vasoactive Mechanisms and Inotropic Action in Disease
]Apelin-13 Identified as the Predominant Apelin Isoform in the Human1[Pyr
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[Pyr1]Apelin-13 Identified as the Predominant Apelin
Isoform in the Human Heart
Vasoactive Mechanisms and Inotropic Action in Disease
Janet J. Maguire, Matthias J. Kleinz, Sarah L. Pitkin, Anthony P. Davenport
Abstract—Apelin receptors, present on vascular smooth muscle cells, endothelium, and cardiomyocytes, are activated by
the family of apelin peptides to elicit cardiovascular effects in experimental animals, but functional activity in humans
has not been studied in detail. We detected low levels of apelin immunoreactivity in plasma of volunteers consistent with
an autocrine/paracrine action and detected apelin immunoreactivity in the supernatant from human cultured endothelial
cells. We found that [Pyr1]apelin-13 was the predominant isoform in cardiac tissue from patients with coronary artery
disease. We tested the hypothesis that apelins have vascular and cardiac actions in human tissues in vitro and compared
responses to [Pyr1]apelin-13, apelin-13, and apelin-36. In endothelium-intact mammary artery, all 3 of the apelins
induced concentration-dependent vasodilatation with comparable potency (EC50: 0.6 to 1.6 nM; maximum response:
40% to 50%). Vasodilatation was abolished after endothelial removal or preincubation with indomethacin but was
unaffected by preincubation with NG-nitro-L-arginine methyl ester, indicating involvement of prostanoids but not NO
in dilatation by apelins in this patient group. Apelins were potent constrictors of endothelium-denuded saphenous vein
(EC50: 0.6 to 1.6 nM; maximum response: 17% to 26%) and mammary artery ([Pyr1]apelin-13; EC50: 0.2 nM; maximum
response: 29%). In paced atrial strips, all 3 of the peptides increased the force of contraction with subnanomolar
potencies (EC50: 40 to 125 pM). For the first time, we demonstrate that the 3 principal forms of apelin have comparable
potency and efficacy in human cardiovascular tissues. Apelins are potent endothelium-dependent vasodilators acting via
a prostanoid-dependent mechanism; however, removal of the endothelium revealed direct vasoconstrictor actions in both
the artery and vein. Furthermore, in human cardiac tissue, the apelin peptides are among the most potent endogenous
positive inotropic agents yet reported. (Hypertension. 2009;54:00-00.)
Key Words: apelin ? human apelin receptor APJ ? vasoconstriction ? vasodilatation
? positive cardiac inotropic agents
A
similarity with the angiotensin II type 1 receptor but does not
bind angiotensin II.1,2The precursor, preproapelin,2–4con-
tains several proteolytic cleavage sites,3predicting formation
of C-terminal peptides, including apelin-36, apelin-13, and
[Pyr1]apelin-13,2,3,5but the endogenous isoforms in humans
remain to be identified. APJ modulates fluid homeostasis4–6
and acts as coreceptor for HIV infection,7,8and apelin is a
novel adipokine9–11with an emerging role in the cardiovas-
cular system.6,12–38A role for apelin in human cardiovascular
disease has been proposed; importantly, mRNA microarray
analysis of ?12 000 genes identified APJ as the most
consistently and significantly increased gene in heart failure
patients after mechanical offloading.12Circulating levels of
apelin may be unchanged13or raised12in early stages of heart
failure but were normalized12or decreased14,15in patients
with severe disease.
pelin peptides, derived from a single gene, activate the
G protein–coupled receptor APJ that has high sequence
Apelin and APJ protein have been detected in endothelial
cells of human arteries and veins,16,17and apelin is a potent
endothelialcellmitogen.18Inhealthyvolunteers,[Pyr1]apelin-13
and apelin-36 produced a rapid increase in forearm blood flow
that was attenuated by NO synthase inhibition but unaffected by
aspirin, implying a role for NO,19presumably released after
activation of endothelial APJ. In rats, apelin lowered blood
pressure,4,5,20and although APJ knockout mice displayed no
change in blood pressure, the pressor response to angiotensin
II was significantly increased.21The action of apelins in the
vasculature of patients with cardiovascular disease has not yet
been studied.
APJ receptors also localized to human and rat vascular smooth
muscle,17,22and we reported previously that [Pyr1]apelin-13 con-
strictedhumansaphenousvein.22We hypothesize that activation
of smooth muscle APJ will produce vasoconstriction that is
counterbalanced by dilatation, mediated by the endothelial
APJ,17in a manner analogous to endothelin 1 (ET-1).39
Received April 15, 2009; first decision May 5, 2009; revision accepted June 11, 2009.
From the Clinical Pharmacology Unit (J.J.M., M.J.K., S.L.P., A.P.D.), University of Cambridge, Centre for Clinical Investigation, Addenbrooke’s
Hospital, Cambridge, United Kingdom.
Correspondence to Janet J. Maguire, Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical Investigation, Box 110,
Addenbrooke’s Hospital, Cambridge, CB2 0QQ United Kingdom. E-mail jjm1003@medschl.cam.ac.uk
© 2009 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.134619
1
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In the heart in vivo, apelin elicited inotropic actions in
rodents23–26thought to result from reflex sympathetic activa-
tion.27However, inotropic effects in a rat-isolated heart17,22
and localization of APJ in rat and human cardiomyocytes17,22
suggested a direct action. In agreement, deletion of the
murine apelin gene resulted in an age-associated reduction in
cardiac contractility.29No studies have yet reported the
cardiac actions of apelins in humans.
Our aim was to identify apelin isoforms in human healthy
and diseased cardiovascular tissues. We detected low levels
in plasma from healthy volunteers and identified [Pyr1]apelin-13
as the predominant isoform in cardiac tissues from patients with
coronary artery disease. We determined the effect of apelin
peptides on vascular tone in endothelium-denuded saphenous
vein and mammary artery and whether they reversed ET-1–
induced vasoconstriction in endothelium-intact artery. We
further delineated the molecular mechanisms underlying
endothelium-dependent vasodilator responses obtained
with [Pyr1]apelin-13 that we detected at highest levels in
these patients. Finally, we sought evidence of a direct
action of apelins on the human isolated paced atria.
[Pyr1]apelin-13 has been identified previously as the most
potent isoform in assays using cells expressing APJ,2,3,30–32
whereas apelin-36 was more effective in inhibiting HIV
infection.8,33,34The rank order of potency of these peptides in
native tissues has not been determined. Therefore, we com-
pared the actions of [Pyr1]apelin-13, apelin-13, and apelin-36.
Methods
All of the experiments described below were in accordance with
institutional guidelines. This investigation conformed to the princi-
ples outlined in the Declaration of Helsinki. An expanded Methods
section is available at http://hyper.ahajournals.org.
Cardiovascular Tissue Collection
All of the samples were obtained with local ethical approval and
informed consent. Human mammary artery (n?46; age: 66.0?1.3
years), saphenous vein (n?43; age: 67.2?1.3 years), and atrial append-
age (n?15; age: 59.9?3.0 years) were from patients receiving coronary
artery bypass grafts, and plasma was from 5 healthy volunteers (age:
29.4?5.7 years). For patient information, see Table S1 in the online
Data Supplement (available at http://hyper.ahajournals.org).
Cell Culture
Human umbilical vein endothelial cells were isolated by collagenase
digestion, grown, and maintained in endothelial cell growth medium
(EBM-2 bullet kit CC-3162; Lonza Wokingham Ltd, Berkshire, UK).
Supernatant was collected from passages 1 to 3 and stored at ?70°C.
Detection of Endogenous Apelin Peptides
Apelin levels were determined in the lyophilized atrial fractions and
plasma and supernatant samples by radioimmunoassay.
In Vitro Functional Studies
Cumulative concentration-response curves were constructed for
[Pyr1]apelin-13, apelin-13, and apelin-36 in endothelium-intact mam-
mary artery preconstricted with ET-1 (10 nmol/L), endothelium-
denuded saphenous vein, and electrically paced atrial appendage strips.
Statistical Analyses
Measurements are mean?SEM. Values of potency (pD2) and max-
imum response (EMAX) were determined using iterative curve-fitting
software (FigP, Biosoft) and compared using 1-way ANOVA with
Bonferroni t test posthoc analysis for multiple comparisons. A
P?0.05 was considered statistically significant.
Results
Measurement of Apelin-Like Immunoreactivity
and Identification of Apelin Isoforms
In plasma from healthy volunteers, we detected low levels of
apelin-like immunoreactivity (0.147?0.011 pmol/L; n?5).
These were below the level for detection of individual isoforms
by high-performance liquid chromatography fractionation. How-
ever, radioimmunoassay detected a single peak of apelin-like
immunoreactivity in the high-performance liquid chromatog-
raphy–fractionated heart extract from patients with cardiovas-
cular disease, corresponding with oxidized [Pyr1]apelin-13
(Figure 1A through 1D; confirmed by mass spectrometry).
Other isoforms were below the level for detection. We
detected significant levels of apelin-like immunoreactivity in
supernatant from human umbilical vein endothelial cells
(0.12?0.03 pmol/L; n?10) compared with blank medium
(?0.04 pmol/L; P?0.05).
Cardiac and Vascular Responses to
Apelin Peptides
Baseline parameters are given in Table S2. In vascular
studies, initial experiments compared [Pyr1]apelin-13, apelin-
13, and apelin-36. Additional mechanistic experiments were
performed using [Pyr1]apelin-13, the major isoform detected
in cardiac tissue from these patients.
Endothelium-Dependent Vasodilator Responses to
Apelin Peptides in Mammary Artery
Constriction to ET-1 was reversed by 40% to 50% by each apelin
peptide with comparable nanomolar potency ([Pyr1]apelin-13 pD2:
8.8?0.1, EMAX: 39?3%, n?12; apelin-13 pD2: 9.2?0.2, EMAX:
51?7%, n?9; apelin-36 pD2: 9.1?0.2, EMAX: 43?6%, n?9;
Figure 2A through 2C). Vasodilator responses to all 3 of the
peptides (n?5) were abolished after endothelium removal (data
shown for [Pyr1]apelin-13; Figure 2D). In endothelium-intact
artery, preincubation with NG-nitro-L-arginine methyl ester
(300 ?mol/L) had no effect (pD2: 9.4?0.4; EMAX: 35?3%;
n?4; Figure 2E), whereas indomethacin (5 ?mol/L) abolished
the [Pyr1]apelin-13 vasodilator response (n?4; Figure 2F).
Controlresponsestoprostacyclininendothelium-denudedartery
elicited a similar maximum response (53?14%; n?6) to
[Pyr1]apelin-13 (39?3%; P?0.05) in endothelium-intact artery
(Figure 3A), and these were significantly less than that of
diethylamine NONOate (115?7%; n?8; Figure 3B). The
control compound diethylamine (n?8) had no effect on
responses to ET-1 (Figure 3B). Dilator responses to
1 ?mol/L of acetylcholine (41.1?8.5% reversal of
10 ?mol/L phenylephrine) were unaffected by NG-nitro-L-
arginine methyl ester (300 ?mol/L; 41.9?6.7%) but atten-
uated by indomethacin (5 ?mol/L; 17.6?8.2%; n?3).
Constrictor Responses to Apelin Peptides in
Saphenous Vein and Mammary Artery
[Pyr1]apelin-13 (pD2: 8.8?0.3; EMAX: 26?4%; n?15),
apelin-13 (pD2: 9.1?0.2; EMAX: 19?5%; n?5), and apelin-36
(pD2: 9.2?0.4; EMAX: 16?3%; n?7) were equieffective con-
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strictors of human endothelium denuded saphenous vein (Fig-
ure 4A through 4C). Responses to [Pyr1]apelin-13 were not
significantly different from responses to angiotensin II (pD2:
8.8?0.2;EMAX: 36?5%KCl;
[Pyr1]apelin-13 was more potent (P?0.05) than phenyleph-
rine (pD2: 6.3?0.2; EMAX: 80?4% KCl; n?10), although it
produced a significantly lower maximum response (P?0.05;
Figure 4D). [Pyr1]apelin-13 also contracted endothelium-
denuded mammary artery (pD2: 9.8?0.6; EMAX: 29?7%
KCl; n?6).
n?10;
P?0.05),and
Positive Inotropic Actions of Apelin Peptides in
Human Paced Atrial Strips
Concentration-dependent increases in the force of contraction,
with comparable subnanomolar potency and efficacy, were
obtained with [Pyr1]apelin-13 (pD2: 9.9?0.2; EMAX: 49?12%;
n?4), apelin-13 (pD2: 10.1?0.3; EMAX: 64?16%; n?4),
and apelin-36 (pD2: 10.4?0.2; EMAX: 39?14%; n?4;
Figure 5A through 5D). These values were more potent
than data we reported previously for ET-1 (pD2: 8.9?0.2;
n?8).40
Figure 1. High-performance liquid chromatography
separation of apelin peptides with detection by
radioimmunoassay for (A) 10 nM mixture of
[Pyr1]apelin-13, apelin-13, and apelin-36 synthetic
peptides; (B) blank run; and (C) atrial appendage
sample (2 g of tissue pooled from n?8); (D) UV
trace showing elution times for standards peptide
mix. Data are expressed as concentration of the
reconstituted fractions in picomoles per liter. From
the graph (C) it can be seen that the oxidized form
of [Pyr1]apelin-13 is present in the atrial sample at
levels that can be detected in this assay.
Maguire et alApelin Actions in Human Cardiovascular Tissue
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Discussion
The predominant vascular effect of apelins in vivo reported in
animals4,21and in healthy human volunteers19is vasodilata-
tion. This study describes for the first time the actions of 3
apelin peptides in cardiovascular preparations from patients
with coronary artery disease. These are in agreement with
previous reports of physiological roles of apelins in the
vasculature4,5,20,35and heart23–25of rodents and with the
presence of apelin receptors in cardiomyocytes, vascular
smooth muscle cells, and endothelial cells.14,17,21,22
It is not known which apelin isoforms have physiological
or pathophysiological relevance. In rats, immunoreactive
apelin was determined to be [Pyr1]apelin-13, apelin-17,41or
apelin-36.31An initial report in human tissues identified apelin
species with molecular weight greater than that of apelin-36,14
although, in a recent study, retention time of immunoreactive
apelin was comparable with apelin-13.13In healthy volunteers
we determined plasma apelin levels to be lower than expected
for a circulating hormone and too low to identify the individual
apelin isoforms. However, we found that, in cardiac tissue from
patients, the predominant isoform was [Pyr1]apelin-13, which
may reflect the protection afforded by the modified pyroglu-
11 10
[Pyr1]apelin-13 [-log mol/L]
9876
60
40
20
0
+ Indomethacin
F
Control
[Pyr1]apelin-13
11 10
[Pyr1]apelin-13 [-log mol/L]
+L-NAME
9876
60
40
20
0
E
* *
** * ** **
*
*
**
Control
[Pyr1]apelin-13
11 10
[Pyr1]apelin-13 [-log mol/L]
9876
60
40
20
0
Endothelium-denuded
D
Control
[Pyr1]apelin-13
11 109876
60
40
20
0
% Reversal of ET-1
[Pyr1]apelin-13 [-log mol/L]
C
A
* **
**
**
**
** ** ** **
11 10
Apelin-13 [-log mol/L]
9876
60
40
20
0
B
*
*
**
*
** ** ** ** **
Control
Apelin-13
11 10
Apelin-36 [-log mol/L]
9876
60
40
20
0
*
**
**
**
** ** ** ** **
Control
Apelin-36
Control
[Pyr1]apelin-13
% Reversal of ET-1
% Reversal of ET-1
Figure 2. Graphs show endothelium-dependent reversal of ET-1
preconstriction in mammary artery by (A) [Pyr1]apelin-13 (F), (B)
apelin-13 (F), and (C) apelin-36 (F). [Pyr1]apelin-13–induced
vasodilatation (F) was abolished in (D) endothelium-denuded
mammary artery and in (F) endothelium-intact vessels pretreated
with indomethacin (5 ?mol/L) but not in (E) endothelium intact
vessels pretreated with NG-nitro-L-arginine methyl ester
(L-NAME; 300 ?mol/L). Data are expressed as mean?SEM.
Open symbols (E) represent measurements from time-matched
controls. Significantly different from time matched control:
*P?0.05, **P?0.005.
87654
120
100
80
60
40
20
0
Agonist [-log mol/L]
B
DEA
DEA/NO
11 10
Agonist [-log mol/L]
9876
120
100
80
60
40
20
0
A
Control
[Pyr1]apelin-13
Prostacyclin
% Reversal of ET-1
Figure 3. Graphs show extent of reversal of ET-1 constriction
by (A) [Pyr1]apelin-13 in endothelium-intact (F) and prostacyclin
in endothelium-denuded mammary artery (f) with time-matched
control (E) and by (B) the NO donor diethylamine NONOate
(DEA/NO; F) and its inactive control diethylamine (DEA; f).
Values are mean?SEM.
11109876
0
10
20
30
40
50
Constriction (%KCl)
[Pyr1]apelin-13 [-log mol/L]
A
11 10
Apelin-13 [-log mol/L]
9876
0
10
20
30
40
50
B
1110987
0
10
20
30
40
50
Apelin-36 [-log mol/L]
C
Constriction (%KCl)
10
Agonist [-log mol/L]
864
0
20
40
60
80
100
D
[Pyr1]apelin-13
Angiotensin-II
Phenylephrine
Figure 4. Vasoconstrictor actions of apelin peptides. Graphs
show vasoconstriction in endothelium-denuded saphenous vein
by (A) [Pyr1]apelin-13, (B) apelin-13, and (C) apelin-36, respec-
tively. Graph in D compares the response to [Pyr1]apelin-13 (F)
with that of angiotensin II (E) and phenylephrine (f). Values are
mean?SEM.
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tamate moiety. These data are consistent with our localization
of apelin-like immunoreactivity to vascular and endocardial
endothelial cells16in this tissue, indicating that apelins may
act as locally released mediators, and, in support of this
hypothesis, we detected apelin-like immunoreactivity in su-
pernatants from human umbilical vein endothelial cells.
In the anesthetized rat in vivo, infused apelin elicited
hypotension, resulting from the generation of NO.20In our
experiments, the vasodilator response to the apelins in human
mammary artery was endothelium dependent, like other
vasoactive transmitters,42but mediated via release of cyclo-
oxygenase products rather than NO. Cyclooxygenase-
mediated apelin signaling has been reported in the rat central
nervous system.43Our observation may reflect species vari-
ation and may be specific to the vascular bed but most likely
reflects endothelial dysfunction in arteries from patients with
cardiovascular disease. This agrees with the only other report
in human isolated blood vessels in which apelin-13–mediated
vasodilatation in mesenteric artery from healthy individuals
resulted from the release of NO, whereas NO was not
involved in the relaxation of hepatic arteries from patients
with liver cirrhosis.36In our study, all 3 of the apelins
reversed ET-1 constriction by 40% to 50%, comparable with
exogenous prostacyclin in endothelium-denuded artery. Thus,
in conditions of endothelial dysfunction where vasoconstric-
tor tone to, eg, ET-1, is enhanced and NO production is
attenuated, apelins have the potential to contribute to benefi-
cial vasodilatation in an NO-independent manner.
Interestingly, in mammary artery, the removal of the
endothelium unmasked vasoconstrictor responses for the
apelins that we also observed in saphenous vein and that were
comparable with responses to angiotensin II. Vascular pep-
tides such as ET-1 elicit both vasodilatation and vasoconstric-
tion; however, for apelins these opposing functions are
mediated via the same receptor, albeit on different cell types
(vascular endothelial cells and smooth muscle cells, respec-
tively). Apelin-mediated phosphorylation of the myosin light
chain kinase.37has been reported in vascular smooth muscle
cells. Thus, in endothelium-intact blood vessels, endothelial
APJ receptors may mediate vasodilatation as a counterregu-
latory mechanism limiting vasoconstriction induced by acti-
vation of APJ expressed on vascular smooth muscle.42
Szokodi et al25demonstrated that apelin was as effective a
positive inotropic agent in the rat heart,25as ET-1,44and as
adrenomedullin,45and we find that the apelins are the most
potent endogenous agents in human atria reported so far.40,46
Apelins have beneficial effects on cardiac performance in
vivo in rats24,26and mice,23and the increase in the cardiac
force of contraction in vitro that we observed suggests that
apelin may also have this potential in humans. Positive
inotropic effects in vivo may be mediated via reflex sympa-
thetic activation.21Our data are the first demonstration of
direct effects of apelins on the force of contraction in human
heart tissue and are consistent with apelin-increased sarco-
mere shortening in rat-isolated cardiomyocytes.38
Variations in relative potencies of apelin peptides have been
reported in cells expressing APJ.2,3,31,32A key finding of our
study is that, in all 3 of the preparations, the relative potencies of
[Pyr1]apelin-13, apelin-13, and apelin-36 were the same; there-
fore, we suggest that APJ activation in the human cardiovas-
cular system will be comparable irrespective of the apelin
isoform involved. This is not unexpected, because it has been
shown that the 12 identical C-terminal amino acids shared by
[Pyr1]apelin-13, apelin-13, and apelin-36 comprise the bio-
logically active core of these peptides.5
Perspectives
Our data show that apelin peptides mediate 3 major actions in
tissues from patients with cardiovascular disease: endotheli-
um-dependent vasodilatation, direct vasoconstriction, and
increased cardiac contractility. Vasodilatation is consistent
with data from animals4,20,21and healthy volunteers.19How-
ever, where the endothelium is dysfunctional in disease, our
studies suggest that, although the balance would be shifted
toward vasoconstriction, some beneficial apelin-mediated
vasodilatation persists that appears to be prostanoid depen-
12 11 10
Apelin-36 [-log mol/L]
987
0
25
50
75
100
C
12 11 10
Apelin-13 [-log mol/L]
987
0
25
50
75
100
B
12 11 10
[Pyr1]apelin-13 [-log mol/L]
987
0
25
50
75
100
Increase in force (% CaCl2
A )
D
10mN
0.01 0.03 0.1 0.3 1 3 10 isoprenaline
0.2mmol/L
••••••••••••••••••••••••••••••••••••••••
1 min
[Pyr1]apelin-13
nmol/L
Figure 5. Inotropic action of apelin pep-
tides. Graphs show increased force of
contraction observed in human paced
atria to (A) [Pyr1]apelin-13, (B) apelin-13,
and (C) apelin-36. D, Representative trace
from a concentration-response curve to
[Pyr1]apelin-13. All of the data are
expressed as mean?SEM.
Maguire et alApelin Actions in Human Cardiovascular Tissue
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dent and NO independent. Data from apelin-infusion studies
in patients is required to confirm this hypothesis. In support,
the hypotensive response to apelin was observed but blunted
in the spontaneously hypertensive rat compared with con-
trols,21and the pressor response to angiotensin II was
augmented in APJ knockout mice.21Blood pressures in the
double angiotensin II type 1A and APJ knockout mice were
raised compared with the angiotensin II type 1A knockout
alone,21indicating that apelin vasodilatation counterbalances
endogenous vasoconstrictor tone, at least in this species. In
patients with chronic heart failure, plasma levels of apelin are
unchanged or increased in early stages12,13but then fall in
more severe disease.12,14,15Our in vitro studies would suggest
that this loss in apelin peptide would result in a concomitant
reduction in beneficial positive inotropic apelin actions and
would promote the deterioration of cardiovascular function.
The development of selective APJ agonists and antagonists in
the future will facilitate basic and integrative research toward
unraveling the role of apelin peptides in the physiology and
pathophysiology of the mammalian cardiovascular system.
Acknowledgments
We thank Jean Chadderton and the theater and consultant staff of
Papworth Hospital for tissue collection.
Sources of Funding
This work was supported by the British Heart Foundation and the
Cambridge European and Isaac Newton Trusts.
Disclosures
None.
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Maguire et alApelin Actions in Human Cardiovascular Tissue
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Online Supplement

[Pyr1]apelin-13 identified as the predominant apelin isoform in
human heart: vasoactive mechanisms and inotropic action in
disease

Janet J. Maguire+, Matthias J. Kleinz+, Sarah L. Pitkin+, & Anthony P. Davenport+

+Clinical Pharmacology Unit, University of Cambridge, Level 6, Centre for Clinical
Investigation, Box 110, Addenbrooke’s Hospital, Cambridge, CB2 0QQ, UK.


Corresponding author: Dr. Janet Maguire
Clinical Pharmacology Unit,
University of Cambridge,
Level 6, Centre for Clinical Investigation,
Box 110, Addenbrooke’s Hospital,
Cambridge, CB2 0QQ, UK.
Phone: (+44) 1223 762579, Fax: (+44) 1223 762576
Email: jjm1003@medschl.cam.ac.uk
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Methods
Cardiovascular Tissue Collection
Human left internal mammary artery (LIMA) (n=46; 37 male, 9 female, mean age 66.0±1.3
years), saphenous vein (n=43; 39 male, 4 female, mean age 67.2±1.3 years) and atrial
appendage (n=15 male, mean age 59.9±3.0 years) were collected with local ethical approval
and informed consent from patients undergoing coronary artery bypass graft surgery. For in
vitro pharmacology, tissues were collected in Krebs solution and maintained at 4°C until use;
for HPLC/radioimmunoassay atrial appendages were frozen and stored at -70ºC until used.
See Table S1 for patient information. Blood was obtained with local ethical approval and
informed consent from 5 healthy volunteers (2 male, 3 female, mean age 29.4±5.7 years).

Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated by collagenase digestion,
grown and maintained in endothelial cell growth media (EBM-2 bullet kit CC-3162). Cell
culture supernatant was collected from passage 1-3 into EDTA coated tubes and stored at -
70ºC.

Detection of Apelin Levels and Isoforms by Radioimmunoassay and HPLC
Extraction of Human Atrial Tissue, Plasma and HUVEC Supernatants
Plasma from 5 individuals, atria from 8 individuals and HUVEC supernatant from 10 cultures
were prepared for solid phase extraction. as described for ET-1.1 Blood samples from 5
healthy volunteers were collected into tubes containing EDTA. Samples were centrifuged at
2,000g for 10 minutes, 4ºC and plasma removed into polypropylene tubes. Atrial tissue
samples (1-2g) from 8 individuals (see Table S1), devoid of fat, were cut into small pieces
and homogenized using a Polytron PT.K homogenizer fitted with a PTA10S probe (Philip
Harris Scientific, Hull, UK) in 10mL/g tissue of 0.5mol/L acetic acid. The homogenate was
heated to 100°C for 15 minutes, cooled and centrifuged at 48000g for 1h, at 4 °C. The
resulting tissue supernatant, plasma and HUVEC supernatant samples were acidified with an
equal volume of 0.1 % trifluoroacetic acid (TFA) and the plasma and HUVEC supernatant
were spun at 2000g for 10 minutes to remove precipitation. All samples were solid phase
extracted using SEP-Pac Vac C18 cartridges containing 500mg of C18 and a vacuum
manifold apparatus. The cartridges were conditioned with 2mL 80% methanol/0.1% TFA and
washed with 9mL 0.1% TFA. Samples were loaded onto the columns and allowed to pass
through under gravity. After washing with 12mL 0.1% TFA the peptides were eluted with
4mL 80% methanol/0.1% TFA. Eluates were evaporated to dryness using a centrifugal
concentrator (Savant Speedvac; Life Sciences International Ltd., Basingstoke, UK) and stored
at –70°C. The percentage recovery of peptide from plasma following solid phase extraction,
determined using a sample of human atrial appendage spiked with apelin-36 (25pg) was 87%.
HPLC analysis
Separations were carried out on a C18 (250×4.60mm) column (Phenomenex, Macclesfield,
UK) using a Gilson 305 binary gradient HPLC system. Flow rate was 1mL/min and 1mL
fractions were collected using a LKB Helirac fraction collector (Pharmacia LKB, St Albans,
Herts, UK). The gradient was selected using 5μmol/L injection of apelin peptides and online
UV detection (Gilson 151 UV/Vis 210 nm). The optimized gradient selected was 20% to
24% acetonitrile with 0.1% trifluoroacetic acid and 0.1% triethylamine over 30 minutes.
Following washing and a blank run, the system was standardized with 10nmol/L injections of
apelin peptides, [Pyr1]apelin-13, apelin-13 and apelin-36. Lyophilized atrial appendage
extracts were reconstituted with deionized water and subjected to the same gradient.
Standards and samples, collected as 1mL fractions, were evaporated to dryness and stored at -
70°C.
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Radioimmunoassay
The lyophilized fractionated atrial tissue and apelin standards, plasma and HUVEC
supernatant samples together with rabbit anti-apelin-36 serum (Phoenix Pharmaceuticals,
Belmont, CA, USA) were reconstituted in assay buffer (50mmol/L sodium phosphate, 0.05 %
Tween 20, 8mmol/L sodium azide, pH 7.4). The biological samples, HPLC fractionated
apelin peptides standards and apelin-36 RIA standards (Phoenix Pharmaceuticals, Belmont,
CA, USA) (100μL samples, appropriately diluted in assay buffer) were incubated with 100μL
of rabbit anti-apelin-36 serum for 16-24 hours at 4 °C. [125I][Pyr1]apelin-13 (Amersham
Biosciences, GE Healthcare UK Ltd, Little Chalfont, UK) at 10 000 counts per minute per
100μL, diluted in assay buffer, was then added and incubated for a further 16-24 hours at 4°C.
Bound tracer was separated from free using Amerlex-M reagent (Amersham Biosciences, GE
Healthcare UK Ltd, Little Chalfont, UK) according to the manufacturers instructions and
bound [125I][Pyr1]apelin-13 in the pellet was detected using a Cobra 5003 gamma counter
(Canberra Packard, Pangbourne, Berks, UK). The standards curves were constructed to
apelin-36 (10-1289pg/mL). The linear portion of the curve was in the range 15-150pg/mL
and IC50 values (the concentration of standard peptide that reduces the binding of the
radioligand by 50%) ranged from 54 to 122pg/mL. The sensitivity of detection was defined
as the amount of standard peptide required to reduce B0 by 2 standard deviations (B0 is
equivalent to 100% [125I][Pyr1]-apelin-13 bound) and was 12pg/mL.
The intra- and inter-assay coefficients of variation (SD/mean) (expressed as a percentage)
were calculated using a sample of human left ventricle from one individual. The intra-assay
coefficient of variation (including variation owing to the solid phase extraction procedure)
was 14.1% (6 determinations); the intra-assay coefficient of variation (excluding extraction
variation, i.e. assay only) was 8.3% (3 determinations). The inter-assay coefficient of
variation was 12.3% over 5 experiments. Cross-reactivity was obtained with [Pyr1]apelin-13
(44%) and apelin-13 (40%)and with the RIA apelin-36 standard (100%). Structurally
unrelated peptides (e.g. angiotensin-II, angiotensin-III, angiotensin-IV, ghrelin, endothelin-1,
bradykinin, motilin, substance P and atrial natriuretic peptide) showed negligible cross-
reactivity in this assay.
We did not detect apelin-like immunoreactivity in the fractions of atrial samples that
coincided with the retention time for apelin-36 on the HPLC column (29 min). For
confirmation, the presence of apelin-like immunoreactivity in these fractions was also looked
for using an additional apelin assay (apelin-12 EIA kit, EK057-23, Phoenix Pharmaceuticals,
Belmont, CA, USA) that showed no significant presence of endogenous apelin-36 in our
sample.
Mass Spectrometry
ESI ms/ms analysis of HPLC fractions was carried out by Dr. L Packman (PNAC Facility,
Dept. Biochemistry, University of Cambridge). Samples were desalted using C18 ziptip and
eluted with 70% MeOH/0.2% formic acid. Samples were analysed on a Thermo Finnigan
LCQ Classic ion-trap mass spectrometer using a static nanospray interface held at 0.7kV.
Tandem ms/ms data was collected manually for target ions. Fragmentation spectra were
interpreted manually and also forwarded as .dta files to Mascot search engine
(www.matrixscience.com) for comparison against the NCBInr database. The peak of
immunoreactivity obtained in the HPLC fractions collected between 8 and 12 minutes in both
the mix of apelin peptides standards and the atrial samples was identified as the oxidized form
of [Pyr1]apelin-13, [Pyr1]Met(O)apelin-13.




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in Vitro Functional Studies
Vascular Dilator Experiments
Histologically normal rings (4mm) of LIMA were left intact or the lumen rubbed gently with
a metal seeker to remove the endothelium2 and mounted in 5mL baths containing oxygenated
Krebs solution for the measurement of isometric force development. Optimum resting tension
was determined by repeated application of 0.1mol/L KCl at increasing levels of basal tension
until no further increase in developed isometric force was obtained. After a 1h equilibration
period, artery rings were pre-constricted with phenylephrine (1µmol/L) and tested for the
presence, or successful removal, of functional endothelium using acetylcholine (1µmol/L).
Those arteries showing a response to acetylcholine of <80% reversal of the phenylephrine
response were not used in subsequent endothelium-dependent studies. A response of <10%
reversal of the phenylephrine response by acetylcholine was required for arterial rings to be
included in the endothelium-denuded studies. Arterial rings were washed and then
preconstricted with ET-1 (10nmol/L) (Peptide Institute, Osaka, Japan), a peptide that
produces sustained vasoconstriction and is known to contribute to the maintenance of vascular
tone.3 Cumulative concentration-response curves were constructed to [Pyr1]apelin-13, apelin-
13 and apelin-36 (10pmol/L-300nmol/L) (Peptide Institute, Osaka, Japan , Phoenix
Pharmaceuticals, Belmont, CA, USA) in the pre-constricted endothelium-intact and denuded
arterial rings with additional control experiments carried out in denuded arterial rings only to
prostacyclin (0.1nmol/L-300nmol/L), the nitric oxide donor diethylamine NONOate
(DEA/NO, Alexis Biochemicals, Nottinghamshire, UK) with diethylamine (DEA) (Alexis
Biochemicals, Nottinghamshire, UK) used as a negative control; both DEA/NO and DEA
used at 10nmol/L-30μmol/L. Experiments were terminated by addition of atrial natriuretic
peptide (ANP, 10nmol/L) followed by S-nitroso-N-acetylpenicillamine (SNAP, 10μmol/L) to
determine maximal receptor mediated and receptor-independent vasodilatation. Adjacent ET-
1 preconstricted rings, to which no apelin peptides were added, served as time-matched
controls.
To investigate the mechanisms involved in any endothelium-dependent responses observed
with the predominant endogenous apelin isoform, concentration-response curves were
repeated to [Pyr1]apelin-13 in endothelium-intact rings pre-incubated with the nitric oxide
synthase inhibitor NG-nitro-L-arginine-methyl ester (L-NAME, 300μmol/L) or the non-
selective cyclo-oxygenase inhibitor indomethacin (5μmol/L).
Vasodilator responses were expressed as percentage reversal of the ET-1 constriction and the
negative logarithm of the EC50 value (pD2) and the maximum response (EMAX) were
determined using iterative curve fitting software (FigP, Biosoft, Cambs, UK).
Vascular Constrictor Experiments
Rings (4mm) of histologically normal saphenous vein and LIMA were mechanically denuded
of their endothelium and mounted in 5mL baths containing oxygenated Krebs solution for the
measurement of isometric force development as described previously4 and above. In
saphenous vein rings at optimized resting tension, cumulative concentration-response curves
were constructed to [Pyr1]apelin-13, apelin-13 and apelin-36 (10pmol/L - 100nmol/L).
Cumulative concentration-response curves were constructed to the established
vasoconstrictors phenylephrine (1nmol/L-100μmol/L) and angiotensin-II (10pmol/L-
100nmol/L) as controls. Experiments were terminated by addition of ET-1 (10nmol/L,
Peptide Institute Inc., Osaka, Japan) followed by KCl (0.1mol/L) to determine maximal
receptor mediated and receptor-independent vasoconstriction. Adjacent rings, to which apelin
peptides were not added, served as time-matched controls. In additional experiments the
predominant endogenous apelin isoform that we detected in cardiac tissue from this patient
group, [Pyr1]apelin-13, was tested for its ability to constrict endothelium-denuded mammary
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artery (4mm rings). Vasoconstrictor responses were expressed as a percentage of KCl
induced vasoconstriction and data analyzed, as above, to determine values of pD2 and EMAX.
Cardiac Functional Studies
Human atrial appendage (n=8) were cut into 3-4 mm strips, parallel to atrial trabecular muscle
bundles and set up in 10mL organ baths containing oxygenated Krebs solution for isometric
tension recordings as previously described.5 After a 1h equilibration period, atrial strips were
paced using field stimulation (1Hz square-wave pulse of 5ms duration, voltage just above
threshold (<4.0V)). Basal tension was incrementally raised until no further increase in force
response was observed. Atrial strips were adjusted to 50% of this tension and left to
equilibrate for 30 minutes. Cumulative concentration-response curves were constructed for
[Pyr1]apelin-13, apelin-13 and apelin-36 (10pmol/L–10nmol/L) and endothelin-1 (ET-1,
Peptide Institute Inc., Osaka, Japan) and experiments were terminated by addition of
isoprenaline (0.2mmol/L), followed by CaCl2 (6.7mmol/L), to determine maximal β1-
adrenoceptor-mediated and external Ca2+-mediated increase in force of contraction. Inotropic
effects of the apelin peptides were expressed as percentage of the maximum increase in force
of contraction for CaCl2 and data analysed to determine values of pD2 and EMAX as described
above.

Statistical Analyses
Measurements were expressed as mean ± standard error of the mean (SEM). Vasodilator
responses are expressed as percentage reversal of the pre-constriction to 10nmol/L ET-1 and
vasoconstrictor responses expressed as a percentage of the terminal response to 0.1mol/L
KCl. Basal parameters are given in Table S2. Values of potency, expressed as the negative
logarithm of the agonist concentration producing 50% of the maximum response (pD2), and
the maximum response (Emax) were determined using iterative curve fitting software (FigP,
Biosoft, Cambs, UK).
Values of Emax, and pD2 for the three apelin peptides were compared using one way analysis
of variance (ANOVA) in combination with Bonferroni’s t-test post hoc analysis for multiple
comparisons. A P value <0.05 was considered statistically significant.

Materials
ET-1 (Peptide Institute Inc., Osaka, Japan) stock solutions (0.1 mmol/L) were prepared in
0.1% acetic acid and stored at -20°C. [Pyr1]apelin-13, apelin-13 and apelin-36 were from the
Peptide Institute, Osaka, Japan, Bachem, St Helens, UK or Phoenix Pharmaceuticals,
Belmont, CA, USA, and were prepared as 1mmol/L stock solutions in water and stored at -
20ºC. Diethylamine NONOate and diethylamine (Alexis Biochemicals, Nottinghamshire,
UK) were stored under nitrogen at -70°C and dissolved in 0.01M NaOH immediately prior to
experiments. Prostacyclin (PGI2) (Sigma-Aldrich Ltd., Dorset, UK) was prepared as a stock
solution (1mM) in distilled water and stored in the dark at 4°C. All other reagents were from
Sigma-Aldrich Ltd. (Dorset, UK) unless stated.
Krebs solution (mmol/L: NaCl, 90; NaHCO3, 45; KCl, 5; MgSO4.7H2O, 0.5; Na2HPO4.2H2O,
1; CaCl2, 2.25; fumaric acid, 5; glutamic acid, 5; glucose, 10; sodium pyruvate, 5; pH 7.4;
gassed with 95% O2/5% CO2, maintained at 37°C).

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References
1. Ashby MJ. Separation of endothelin isoforms by high performance liquid
chromatography and detection by radioimmunoassay. Methods Mol Biol. 2002;206:37-
42.
2. Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary
artery: ghrelin is a potent physiological antagonist of endothelin-1. Br J Pharmacol.
2002;136:1146-1152.
3. Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal
vascular tone. Lancet. 1994;344:852-854.
4. Katugampola SD, Maguire JJ, Matthewson SR, Davenport AP. [(125)I]-(Pyr(1))Apelin-
13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues
with evidence for a vasoconstrictor role in man. Br J Pharmacol. 2001;132:1255-1260.
5. Sandilands AJ, O'Shaughnessy KM, Brown MJ. Greater inotropic and cyclic AMP
responses evoked by noradrenaline through Arg389 beta 1-adrenoceptors versus Gly389
beta 1-adrenoceptors in isolated human atrial myocardium. Br J Pharmacol.
2003;138:386-392.

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Table S1. Patient Information
Mammary artery from 46 patients, 37 male, 9 female, mean age 66.0± ±1.3 years
Age (years) Sex Drug treatment before CABG surgery
69 F
Aspirin, Metoprolol, Ramipril, ISMN, GTN, Nicorandil, Frusemide,
Simvastatin, Temazepam, Coproxamol, Lansoprazole
57 M Aspirin, Enoxaparin, Doxazosin, Ramipril, Amlodipine, Frusemide,
Amiloride, Atorvastatin, Paracetamol, Tramadol, Metformin, Gliclazide,
Flucloxacillin
65 M Digoxin, Verapamil, Frusemide, Simvastatin
79 M Aspirin, Atenolol, Ramipril, GTN, Nicorandil, Simvastatin, Metformin
70 M Details not available
76 M Details not available
64 M Aspirin, GTN, Nicorandil, Amiloride, Atorvastatin
69 M Aspirin, Ramipril, Diltiazem, Nicorandil, Simvastatin, Metaformin,
Gliclazide
69 M Valsartan, GTN, ISMN, Frusemide
78 M Aspirin, Lisinopril, GTN, Nicorandil, Frusemide, Atorvastatin
71 M Aspirin, Atenolol, ISMN, GTN, Atorvastation, Paracetamol
66 F
Bisoprolol, Ramipril, Nicorandil, Atorvastatin, Paracetamol, Panadol,
Tramadol
69 M Aspirin, Nicorandil, Bendroflumethazide, Tramadol, Coproxamol,
Diclofenac, Salbutamol, Zopiclone
65 M Aspirin, Atenolol, GTN, Atorvastatin
69 F
Aspirin, Atenolol, Nifedipine, ISMN, Nicorandil, Atorvastatin,
Coproxamol, Thyroxine
74 F Aspirin, Atenolol, Atorvastatin, Paracetamol
57 M Aspirin, Clopidogrel, Atenolol, Perindopril, GTN, Rosuvastatin
55 M Details not available
67 M Aspirin, Atenolol, Amlodipine, GTN, Simvastatin
59 M Clopidogrel, Bisoprolol, Verapamil, Nicorandil, Simvastatin
72 M Aspirin, Bisoprolol, Lansoprazole, Ferrous sulphate
62 M Aspirin, Atenolol, Ramipril, Amlodipine, Nicorandil, Bendrofluazide,
Atorvastatin
62 M Aspirin, Bisoprolol, Ramipril, Simvastatin, Thiamine, Ferrous sulphate
77 M Aspirin, Clopidogrel, Enoxaparin, Bisoprolol, Furosemide, Simvastatin
64 F
Aspirin, Clopidrogrel, Metoprolol, GTN, ISMN, Furosemide,
Atorvastatin,
64 M Warfarin, Sotalol, Simvastatin, Omeprazole, Lactulose
67 F Aspirin, Ramipril, Diltiazem, ISDN, Atorvastatin, Omeprazole
59 F Aspirin, Atenolol, ISMN, GTN, Atorvastatin
73 M Aspirin, Bisoprolol, Atorvastatin
75 M Aspirin, Atenolol, GTN, Atorvastatin
72 M Perindopril, Simvastatin, Alendronate
59 M Aspirin, Bisoprolol, Telmisartan, GTN, Nicorandil, Simvastatin,
Temazepam

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