Growth-hormone-releasing peptide 6 (GHRP6) prevents oxidant cytotoxicity and reduces myocardial necrosis in a model of acute myocardial infarction.

Abstract

Therapies aimed at enhancing cardiomyocyte survival following myocardial injury are urgently required. As GHRP6 [GH (growth hormone)-releasing peptide 6] has been shown to stimulate GH secretion and has beneficial cardiovascular effects, the aim of the present study was to determine whether GHRP6 administration reduces myocardial infarct size following acute coronary occlusion in vivo. Female Cuban Creole pigs were anaesthetized, monitored and instrumented to ensure a complete sudden left circumflex artery occlusion for 1 h, followed by a 72 h reperfusion/survival period. Animals were screened clinically before surgery and assigned randomly to receive either GHRP6 (400 microg/kg of body weight) or normal saline. Hearts were processed, and the area at risk and the infarct size were determined. CK-MB (creatine kinase MB) and CRP (C-reactive protein) levels and pathological Q-wave-affected leads were analysed and compared. Evaluation of the myocardial effect of GHRP6 also included quantitative histopathology, local IGF-I (insulin-growth factor-I) expression and oxidative stress markers. GHRP6 treatment did not have any influence on mortality during surgery associated with rhythm and conductance disturbances during ischaemia. Infarct mass and thickness were reduced by 78% and 50% respectively, by GHRP6 compared with saline (P<0.01). More than 50% of the GHRP6-treated pigs did not exhibit pathogological Q waves in any of the ECG leads. Quantitative histopathology and CK-MB and CRP serum levels confirmed the reduction in GHRP6-mediated necrosis (all P<0.05). Levels of oxidative stress markers suggested that GHRP6 prevented myocardial injury via a decrease in reactive oxygen species and by the preservation of antioxidant defence systems (all P<0.05). Myocardial IGF-I transcription was not amplified by GHRP6 treatment compared with the increase induced by the ischaemic episode in relation to expression in intact hearts (P<0.01). In conclusion, GHRP6 exhibits antioxidant effects which may partially contribute to reduce myocardial ischaemic damage.

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Clinical Science (2007) 112, 241–250 (Printed in Great Britain)doi:10.1042/CS20060103
241
Growth-hormone-releasing peptide 6 (GHRP6)
prevents oxidant cytotoxicity and reduces
myocardial necrosis in a model of acute
myocardial infarction
Jorge BERLANGA∗, Danay CIBRIAN∗, Luis GUEVARA†, Heberto DOMINGUEZ‡,
Jose S. ALBA∗, Alina SERALENA∗, Gerardo GUILL´EN∗, Ernesto L´OPEZ-MOLA∗,
Pedro L´OPEZ-SAURA∗, Alberto RODRIGUEZ§, Brumny PEREZ∗, Diana GARCIA∗
and Nelson S. VISPO∗
∗Center for Genetic Engineering and Biotechnology, Ave. 31 e/ 158 and 190, P.O. Box 6162, Cubanac´ an, Playa, Havana,
Cuba, †Department of Cardiology and Cardiovascular Surgery, Hermanos Ameijeiras Hospital, Avenida Antonio Maceo, esquina
Belascoa´ ın S/N, Centro Habana, Havana, Cuba, ‡Department of Surgery, Swine Research Institute, Carretera del Guatao,
Punta Brava, La Lisa, Havana, Cuba, and §Cardiology Devices Service, Institute for Digital Research, 202 St. Siboney, Playa,
Havana, Cuba
ABSTRACT
Therapies aimed at enhancing cardiomyocyte survival following myocardial injury are urgently
required. As GHRP6 [GH (growth hormone)-releasing peptide 6] has been shown to stimulate GH
secretion and has beneficial cardiovascular effects, the aim of the present study was to determine
whether GHRP6 administration reduces myocardial infarct size following acute coronary occlusion
in vivo. Female Cuban Creole pigs were anaesthetized, monitored and instrumented to ensure a
complete sudden left circumflex artery occlusion for 1 h, followed by a 72 h reperfusion/survival
period. Animals were screened clinically before surgery and assigned randomly to receive either
GHRP6 (400 µg/kg of body weight) or normal saline. Hearts were processed, and the area at risk
and the infarct size were determined. CK-MB (creatine kinase MB) and CRP (C-reactive protein)
levels and pathological Q-wave-affected leads were analysed and compared. Evaluation of the
myocardial effect of GHRP6 also included quantitative histopathology, local IGF-I (insulin-growth
factor-I) expression and oxidative stress markers. GHRP6 treatment did not have any influence on
mortality during surgery associated with rhythm and conductance disturbances during ischaemia.
Infarct mass and thickness were reduced by 78% and 50% respectively, by GHRP6 compared with
saline (P<0.01). More than 50% of the GHRP6-treated pigs did not exhibit pathogological Q
waves in any of the ECG leads. Quantitative histopathology and CK-MB and CRP serum levels
confirmed the reduction in GHRP6-mediated necrosis (all P<0.05). Levels of oxidative stress
markers suggested that GHRP6 prevented myocardial injury via a decrease in reactive oxygen
species and by the preservation of antioxidant defence systems (all P<0.05). Myocardial IGF-I
transcription was not amplified by GHRP6 treatment compared with the increase induced by
the ischaemic episode in relation to expression in intact hearts (P<0.01). In conclusion, GHRP6
exhibits antioxidant effects which may partially contribute to reduce myocardial ischaemic damage.
Keywords:cardioprotection,growth-hormone-releasingpeptide6(GHRP6),infarction,ischaemia,myocardium,necrosis,oxidative
stress.
Abbreviations: AAR, area at risk; AMI, acute myocardial infarction; BMS, biomodel standardization ; CK-MB, creatine kinase-
MB; CRP, C-reactive protein; GH, growth hormone; GHRP, GH-releasing peptide; I/R, ischaemia/reperfusion; IGF-I, insulin-like
growth factor-I; LV, left ventricular; MDA, malondialdehyde; NBT, Nitroblue Tetrazolium; PI3K, phosphoinositide 3-kinase; ROS,
reactive oxygen species; RT, reverse transcriptase; SODt, total superoxide dismutase; T0, basal pre-ischaemia; T1, at 30 min after
the beginning of the reperfusion period; T2, the end of 72 h of reperfusion following ischaemia; THP, total hydroperoxides; WBC,
white blood cell.
Correspondence: Dr Jorge Berlanga (email jorge.berlanga@cigb.edu.cu).
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INTRODUCTION
AMI (acute myocardial infarction) is the rapid onset
of myocardial necrosis caused by a sudden critical im-
balance between oxygen supply and its demand by the
myocardium [1]. As cardiac myocytes are terminally dif-
ferentiatedcells,theirabilitytoregenerateisverylimited,
thus the consequences of I/R (ischaemia/reperfusion)
damage within the injured myocardium can only be
partiallyrepaired[2].Thepreservationofmyocardialand
other tissue viabilities during and following an ischaemic
episode is therefore a major goal for modern medicine.
GHRPs [GH (growth hormone)-releasing peptides]
are a heterogeneous group of synthetic peptidic agents
that act as potent GH secretagogues via specific G-
protein-coupled receptors in the hypothalamus and the
pituitary [3]. Different studies indicate that GHRP6 and
hexarelin exert potent cardioprotective actions, includ-
ing in models of myocardial injury by ischaemia or
hypoxia [4–6]. Some of these experimental findings were
substantiated further in clinical studies which demon-
strated that LV (left ventricular) ejection fraction, cardiac
index and cardiac output were improved upon hexarelin
administration[7,8].Furthermore,Iwaseetal.[9]recently
demonstrated that GHRP6 attenuates LV dysfunction
and dilation in hamsters with cardiomyopathy. Funda-
mental to the present study is the finding that GHRP6
treatment prevented sudden death in dogs with cardio-
myopathy subjected to AMI, without reducing infarct
size or ameliorating ischaemia-associated electrical in-
stability [10]. Previous observations from our group [11]
have indicated that GHRP6 exerts potent cytoprotective
effects by enhancing tissue viability in acute I/R episodes
in different splanchnic organs (small bowel, liver and
kidneys). However, the mechanisms whereby GHRP6
enhances cell tolerance to otherwise lethal insults such
as hypoxia remain to be fully understood. Our own
observationsledustosuggestthehypothesisthatGHRP6
may also assist in myocardial tissue survival during an
ischaemic episode. The main aim of the present study
was to determine whether pharmacological intervention
with GHRP6 in a combined scheme of administration
contributed to rescue cardiomyocytes from necrosis and
thus reduce infarct size in a porcine model of acute
coronary occlusion. The present findings suggest that the
pro-survivaleffectofGHRP6isrelatedtotheattenuation
of I/R-associated oxidative cytotoxicity.
MATERIALS AND METHODS
Animals
Female inbred Cuban Creole pigs weighing 23–29 kg
wereused.AnimalswereprovidedbytheNationalSwine
Genetic Center, Havana, Cuba. Body weight during the
study was ensured by the stepwise purchase of litter-
mates of approx. ten pigs. They were maintained and
manipulated in accordance with the Guide for the Care
and Use of Laboratory Animals of the National Swine
Research Institute. Animals were fasted for 16 h before
surgery.
GHRP6
The hexapeptide GHRP6 (His-d-Trp-Ala-Trp-d-Phe-
Lys-NH2) was purchased from BCN Peptides (pyrogen-
free with 95% purity). The peptide was aliquoted into
sterile vials in a laminar flow hood and kept at −20◦C
untiluse.Forinvivoadministration,freshsolutionswere
always prepared by diluting the peptide in sterile normal
saline.
Standardization of the animal model
For inclusion in the study, all of the pigs from each litter
were subjected to a clinical assessment, which included
bodyweightandtemperature,heartandrespiratoryrates,
12-leadECG,haemoglobin,totalWBC(whitebloodcell)
count, and basal levels of CK-MB (creatine kinase-MB)
and CRP (C-reactive protein). These examinations were
done during the acclimitization period approx. 7–9 days
prior to surgery. A non-lethal infarct model was required
in order to achieve an appropriate survival window for
the study. Sixteen animals were assigned to the biomodel
standardization (BMS) group in order to assess the
feasibilityofinducingarepeatableandreproducibleAMI
in the pig strain used. Left circumflex artery clamping
was the preferred procedure. An ischaemic period of
60 min was used as reported in a previous study [10].
Animals were monitored electrocardiographically before
surgery, during the acute I/R period and once a day
until completing a 72 h reperfusion period. After this
period, the pigs were killed. The pilot studies revealed
that 72 h was an appropriate time window to determine
myocardial changes. Myocardial injury was studied as
described below.
Anaesthesia and surgery
Pigs were anaesthetized with a combination of ketamine
(16 mg/kg of body weight), flunitrazepam (0.45 mg/kg
of body weight) and pavulon (0.03 mg/kg of body
weight) via a tube inserted into an ear marginal vein. A
cuffed endotracheal tube was inserted, and the animals
were ventilated with a Mark 10 machine. Animals were
monitored electrocardiographically using a 12-lead solid
state CYS SG-electrocardiographic device (ICID) sup-
plied with CARDIOCID PC (EICISOFT) software,
allowing for continuous on-screen ECG and heart rate
registers, and for automatic recordings every 20 s. A left
thoracotomy was performed between the fourth and
fifth intercostal spaces. Once the heart was exposed,
a cavafix-indwelling catheter was inserted into the left
atrium and its syringe connection tip was externalized
between the scapulae. After opening the pericardium, the
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GHRP6 reduces myocardial ischaemic necrosis
243
coronary branches were dissected and the left circumflex
artery was exposed, suspended and totally occluded for
1 h with a vascular bulldog clamp (Fine Science Tools).
Once the ischaemic period was completed, the clamp was
removed and the chest was sutured in layers.
Allocation to the experimental groups,
GHRP6 administration and doses
Once the baseline clinical assessment and the blood
chemistry data were reviewed, 36 pigs were randomly
assigned to one of the two groups with 18 animals in
each: group I, saline placebo, or group II, GHRP6 treat-
ment. Allocation was done using a computer-generated
randomization list, matching the animal number to either
group I or II. A balanced-group distribution was con-
firmed thereafter by reviewing the baseline data, and no
pigs were excluded. Nine animals survived in each group
until being killed at 72 h post-coronary occlusion. Pigs
assigned to group I received 10 ml of sterile normal saline
equilibrated at 37◦C. For group II, GHRP6 was admin-
istered at a dose of 400 µg/kg of body weight in an
equal volume of normal saline. The treatments were
infused via a cavafix catheter. The dosing regimen used
was introduced to investigate the potential influence
of the acute treatment on specific myocardial electrical
derangements (detected by electrocardiography) associ-
ated with different conditions during I/R. GHRP6 inter-
ventions were as follows. (i) A pre-ischaemia bolus, given
10 minbeforecircumflexarteryocclusion,withacuteST-
segment elevation being the parameter observed. (ii) A
second bolus was infused 20 min after clamping, coincid-
ing with the peak of arrhythmic beats and ventricular
fibrillation, to examine whether GHRP6 ameliorated
ischaemia-associatedventriculararrhythmias.(iii)Athird
bolus was infused 5 min before the beginning of the
reperfusion period in order to characterize reperfusion
arrhythmias. Treatments were continued thereafter twice
a day at regular intervals. Animals were killed at 72 h
aftersurgery.TheGHRP6doseusedinthisstudyderived
from our liver I/R dose–response experiments in which
100 and 400 µg/kg of body weight exhibited similar
hepatoprotective effects (D. Cibrian and J. Berlanga, un-
published work). The 400 µg dose was preferred because
it was twice the dose reported to prevent sudden death in
dogs with cardiomyopathy subjected to acute I/R with
no resulting differences in infarct size [10]. Furthermore,
such a dose is supported by pharmacokinetic and bio-
distribution data in male Wistar rats (J. Berlanga and
D. Cibrian, unpublished work), which demonstrated a
half-life of 12 h (90% of the area under the curve) and
a myocardial residence of 0.2% after 24 h.
Infarct size and weight
A few minutes after clamping the circumflex artery,
200 ml of 2.5% (w/v) Evans Blue solution was infused.
The unstained epicardial territory was traced on a trans-
parent sheet and digitally processed further to determine
the AAR (area at risk). These data were used to calcul-
ate the percentage of the infarcted area in relation to the
original myocardial AAR. After 72 h of reperfusion,
the pigs were anaesthetized and a final 12-lead ECG re-
cording was done. Again, 2.5% (w/v) Evans Blue sol-
ution was infused to assist in delimiting the necrotic
area, particularly when it was small. Subsequently, the
hearts were arrested with 0.5 mmol/l KCl, excised and
processed to determine infarct size, as described pre-
viously [12,13]. Heart weights were recorded. Infarct
thickness was measured with a caliper square along the
affected ventricular wall after a longitudinal incision at
three different points to obtain average values. Thus the
average value of the wall necrosis thickness per group
is reported. Transverse sections were taken on the LV
wall from the apex upwards to the level just below the
coronary sulcus. Serial sections were then incubated in
1% NBT (Nitroblue Tetrazolium; Sigma-Aldrich) for
20 min at 20◦C, and then immersed in 10% buffered
formalin [12]. After NBT incubation, the area of viable
perfused tissue was coloured blue, viable ischaemic tissue
was coloured red (stunned myocardium) and necrotic
tissue appeared pale or white. The necrotic territory was
excised, weighed and its weight adjusted to the totalheart
weight to determine the percentage of the infarcted mass.
In addition, the percentage of the infarcted area was also
given adjusted to the AAR [13].
Myocardial tissue characterization
In order to characterize the impact of treatment on the
biochemical and structural preservation of myocardial
tissue, standard-sized transmural punch biopsies were
harvested from macroscopically normal myocardium
(Acupunch biotomes; Acuderm) adjacent to the necrotic
core. Tissue fragments were used to examine myocardial
expression of IGF-I (insulin-like growth factor-I), to
measure oxidative damage and for histopathological
analysis. Five additional hearts samples were obtained
fromsex-andage-matchedhealthypigsofthesamestrain,
whichwereusedasareferenceforconstitutiveexpression
of IGF-I and to gain information on basal myocardial
redox status in an intact control group.
Myocardial transcriptional expression of IGF-I was
examined using RT (reverse transcriptase)-PCR. Total
RNA was isolated from intact hearts and from those
subjected to I/R and receiving either saline or GHRP6
(n=5 in each group), using TRIzol (Invitrogen). Total
RNA was digested with RNase-free DNase I (Epicentre
Technologie), according to the manufacturer’s instruc-
tions. A total of 3 µg of RNA were reverse-transcribed
using the GeneAmp®RNA PCR Core kit (Applied
Biosystems) with an oligo(dT) primer. PCR was per-
formed using the following porcine IGF-I-specific
primers: 5?-CTGTGGGGCTGAGCTGGTGGACG-3?
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(sense) and 5?-AAGGATCCTGCCAGTGGCATGTC-
3?(antisense) (GenBank®accession number M31175). A
final product of 353 bp was generated after 30 cycles at
68 and 94◦C. IGF-I expression was normalized against
porcine β-actin expression as a housekeeping gene using
the following primers: 5?-GGAGATCGTGCGGGAC-
ATCAAGG-3?(sense) and 5?-GGCCGGACTCGTC-
GTACTCCTGC-3?(antisense) (Genbank®accession
number AY 550069). After 30 cycles at 68 and 94◦C,
the amplification process produced a final product of
482 bp. The bands were detected in a 1% (w/v) agarose
gel and were quantified using the Kodak I D 3.6 software
package.
For assessment of lipoperoxidative damage, tissue
homogenates [1:10 (w/v)] were prepared by myocardial
disruption in 50 mmol/l KCl/5 mmol/l histidine buffer
(pH 7.4) at 4◦C using a tissue homogenizer (T25 Basic;
IKA Labortechnik) for 3 min at 15000 rev./min and were
subsequently centrifuged at 5000 g at 4◦C for 20 min
(HimacSCR20B;Hitachi).TissueaccumulationofMDA
(malondialdehyde) and THP (total hydroperoxides), as
well as SODt (total superoxide dismutase) and catalase
activities, were determined in the supernatants. All of the
biochemical parameters were measured by spectropho-
tometric methods using an Ultrospec 2000 UV/Visible
spectrophotometer (Pharmacia Biotech). MDA and
THP were determined using the Bioxytech LPO-586
and Bioxytech H2O2-560 kits respectively, according
to manufacturer’s instructions (Bio-Rad Laboratories).
SODt activity was estimated by determining the capacity
of the enzyme to inhibit the auto-oxidation of pyrogallol
at a rate of 50% [14]. Catalase activity was based
on the reduction of H2O2 to oxygen and water, as
described previously [15]. All of the biochemical data
were adjusted to total protein concentration determined
inthetissuesupernatantsusingacommercialkit(Bio-Rad
Laboratories).
For histopathological analysis, normal and damaged
myocardial samples were harvested and fixed in 10%
(v/v) formalin, paraffin-embedded and processed for
haematoxylin/eosin and basic fuschin staining. LV wall
integrity was determined qualitatively by two independ-
ent pathologists. In addition, quantitative parameters,
such as the number of necrotic peninsulas within the
tissue section and the total number of irreversibly
damaged nuclei (exhibiting karyorrhexis, karyolysis
and/or karyopyknosis) in 10–15 microscopic fields at
×10–20 magnification, were examined in serial sections,
as described previously [16]. All of the pathological
studies were performed in a blinded manner.
ECG study
The time points at which rhythmic and conductance
disturbances started, including ST elevation, ventricular
arrhythmic beats and ventricular fibrillation, were
registered in each ECG during the ischaemic period.
Table 1
groups
Values are means+−S.D. Animals were screened clinically before surgery and study
group allocation to ensure that they were healthy and that the experimental
arms were suitably balanced. No significant differences were detected (P >0.05)
between the groups, as determined by an unpaired Student’s t test.
Baseline clinical characterization of the study
Group
ParameterBMSSalineGHRP6
Body weight (kg)
Heart rate (beats/min)
Respiratory rate
(breaths/min)
Haemoglobin (g/l)
WBC (×109/l)
CK-MB (units/l)
CRP (µg/ml)
25.66+−1.97
73+−4
44+−3
123.84+−7.73
8+−1.41
1267.72+−56.18 1270.23+−43.25 1288.55+−81.97
0.087+−0.004
27.62+−2.41
75+−6
42+−4
120.56+−6.44
9+−2.17
0.09+−0.0026
26.31+−2.25
74+−7
46+−6
121.77+−7.03
8+−1.66
0.093+−0.0041
Duration of reperfusion arrhythmia and the time point
of the resumption of sinusal rhythm within the acute
reperfusion phase were also determined individually.
Both basal and final (pre-autopsy) ECG recordings were
analysed and compared to determine the number of leads
showing pathological Q waves in each animal.
Circulating levels of CK-MB and CRP
Circulating CK-MB and CRP levels were determined
in serum collected before surgery (T0), 30 min after the
beginningofthereperfusionperiod(T1;atchestsuturing)
andattheendof72 hofreperfusion(T2;beforeautopsy).
Commercially available kits were used to determine CK-
MB (Roche Diagnostics) and CRP (Diagnostics Auto-
mation), according to the manufacturers’ instructions.
However, results should be interpreted with caution as
thekitsusedareintendedforhuman,ratherthanporcine,
samples.
Statistical analysis
All of the experimental data were initially evaluated for
a normal distribution using the Kolmogorov–Smirnov
test (P<0.05). When a normal distribution was estab-
lished, an unpaired Student’s t test was used, except
for oxidative-stress-related parameters in which one-
way ANOVA followed by the Student–Newman–Keuls
multiple comparisons test was used. A P value <0.05 was
used to indicate a significant difference.
RESULTS
Animal model
The baseline clinical characterization of the animals is
summarized in Table 1. The similarity of the values indi-
cated that the experiment was done on healthy animals,
that no clinical differences existed among the groups
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245
Table 2
groups
Values are means+−S.D. No statistical differences were detected when heart
weights were compared among the groups (P =0.81), indicating an appropriate
allocation of animals.
compared with the saline group, as determined by an unpaired Student’s
t test.
Heart weights and infarct dimensions in the study
∗∗P ?0.01,
∗∗∗P ?0.001 and
∗∗∗∗P <0.0001
Group
Parameter BMS SalineGHRP6
Heart weight (g)
Infarct mass (absolute
weight in g)
Infarcted mass (%)
Infarcted area (cm2)
AAR (cm2)
Infarcted area/AAR (%) 77.89+−18.17 74.51+−23.82 15.33+−14.78∗∗∗∗
Infarct thickness (cm)1.58+−0.52
158.23+−11.64 156.18+−15.45 153.92+−26.2
39.4+−10.55
24.91+−4.78
32.06+−12.27 30.31+−14.74
40.44+−6.77
1.56+−0.30
37.4+−11.81 12.03+−10.09∗∗∗
24.0+−7.24
38.78+−8.03
7.81+−5.63∗∗
6.46+−6.25∗∗
39.67+−4.80
0.7+−0.61∗∗
Table 3
study groups
Values are means+−S.D. of the time point at which the electrophysiological
changes started and were registered along the continuous ECG automatic recording.
No significant differences were detected (P >0.05) between the groups, as
determined by an unpaired Student’s t test.
Electrocardiographic changes during I/R in the
Group
ParameterBMSSaline GHRP6
ST elevation (s)
Ventricular arrhythmias (min)
Ventricular fibrillation/
sudden death (min)
Sinusal rhythm resuming (min)
54+−3.14
18.54+−11.15 17.36+−10.37 19.75+−13.82
17.24+−12.22
5.68+−4.97
53+−2.61
15.6+−13.16
5.89+−5.69
58+−5.30
20.8+−5.43
4.56+−5.18
and that the experimental arms of the study were bal-
anced. Ventricular fibrillation/sudden death, appearing
during the ischaemic phase, led to the death of 50% of
thepigsusedduringsurgicalprocedureintheBMSgroup
as in the study groups. Fibrillation frequently started
after15–20 minofclampingtheleftcircumflexarteryand
could be readily anticipated by extrasystolic ventricular
beats. The remaining 50% of animals that tolerated the
ischaemic impact had uneventful post-surgical events.
The infarcts appeared limited to the lateral side of the
left ventricle and were largely similar in terms of size and
depth (Table 2). Hearts were also largely similar in terms
of electrocardiographic changes (Table 3).
Effect of GHRP6 on infarct dimensions
As shown in Table 2, mean heart weights were very
similar between the two groups involved in the study
(P=0.81). Treatment with GHRP6 significantly reduced
Figure 1
Q waves
Pathogological Q waves representative of myocardial necrosis were identified in
ECG leads. Pre-autopsy ECGs, representing 72 h of post-occlusion reperfusion,
were studied and compared with basal and during-surgery ECG recordings. A
significant difference (P <0.001) was observed between the saline and GHRP6
groups, as determined by an unpaired Student’s t test.
Effect of GHRP6 on the incidence of pathogological
the absolute weight of the infarct territory compared
with the saline controls (P=0.001). Consequently, the
infarctmassadjustedtothetotalheartweightwasreduced
by 78% by GHRP6 treatment (P=0.005). There were
no statistical differences in the AAR between the three
groups (P=0.78), indicating that our results were not
influenced by collateral networks. This may explain why,
when the infarcted area was adjusted to the value of the
myocardial AAR, infarct reduction remained close to
80% (P<0.0001). Accordingly, infarct size and thick-
ness appeared significantly reduced (P=0.005) in the
GHRP6-treated animals.
Effect of GHRP6 on myocardial electrical
instability
GHRP6 treatment did not prevent or delay the onset
of acute rhythmic and conductance disturbances during
the ischaemic episode or those associated with the early
reperfusion period. Consequently, mortality during the
surgicalprocedureassociatedwithventricularfibrillation
was not reduced by GHRP6 intervention. As shown in
Table3,thetimepointsatwhichSTelevation,arrhythmic
ventricular beats and ventricular fibrillation/sudden
death started were very similar between the groups (all
P>0.05). In the reperfusion phase, GHRP6 treatment
did not prevent or reduce the duration of reperfusion ar-
rhythmias(P>0.05).IrrespectiveofthefactthatGHRP6
treatment did not ameliorate myocardial electrical
instability, the ECG study confirmed its benefits in terms
of a reduction in myocardial necrosis, as judged by
the number of ECG leads showing abnormal Q waves.
Figure 1 shows that five out of nine animals treated
with GHRP6 did not exhibit pathological Q waves in
any of the 12 leads studied. In contrast, pathological Q
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Figure 2
(A and B) Macroscopic assessment. (A) In the saline group, extensive transmural coagulative necrosis of the left ventricle was seen. (B) In the GHRP6 group, a small
infarcted area was observed (arrow indicates small apexian infarct). (C and D) Histopathological assessment of transmural punch biopsies (6 mm diameter) retrieved
from apparently normal areas close to the myocardial injury core. Tissue samples were fixed in formalin, paraffin-embedded, sectioned into 5 µm slices and stained
with haemotoxylin/eosin. (C) A representative photomicrograph of myocardial tissue from the saline group showing myofibrolysis, inflammatory cell infiltration and
myofibres within the field (magnification, ×20). (D) A representative photomicrograph of myocardial tissue from the GHRP6 group showing that that myofibres were
mostly preserved or only mildly damaged (sarcoplasmic oedema). No myofibrolysis was observed, although a number of ghost nuclei appeared (magnification, ×20).
Effect of GHRP6 on the morphological characterization of the myocardium
wavesappearedinallofthesaline-treatedanimals,mostly
involving four or five ECG leads (P<0.001).
Myocardial morphological characterization
Necrosis was the most prominent form of myocardial
death within the damaged area, as determined by NBT
stainingandhistopathology.Areassuggestingmyocardial
stunning were rarely detected. The necrotic core ap-
peared as a white well-delimited zone (Figure 2A), pre-
dominantly involving the lateral side, in agreement with
the AAR following Evans Blue staining. Treatment
withGHRP6resultedingrossdifferencesinmorphology
compared with the controls (Figure 2B). Histological
examinationofthetransmuralbiopsiesdemonstratedthat
substantial differences existed in relation to fibre lysis,
inflammatory infiltrate intensity, haemorrhage extension
and nuclear viability in areas close to the infarcted core
betweenthecontrol(Figure2C)andGHRP6(Figure2D)
groups. As shown in Figure 2(D), preservation of fibre
integrity was a major hallmark of the effect of GHRP6
treatment compared with the predominant lytic pattern
found in saline-treated animals (Figure 2C). Inflam-
matory infiltrate and haemorrhagic events appeared to
be attenuated more in the samples from the GHRP6
group. Furthermore, myocardial necrotic peninsulas
and the amount of lethally injured nuclei appeared
significantly reduced (P<0.001) in the GHRP6-treated
animals compared with the controls (Table 4).
Serum CK-MB and CRP levels
The changes observed in CK-MB levels suggested that
GHRP6preservedmyocardialviability.AsshowninFig-
ure3(A),asignificantincreasewasdetectedinthecontrol
group at T1 (at 30 min after the beginning of the reper-
fusion period) compared with T0 (basal pre-ischaemic
values; P<0.01). A 5-fold increase was observed in the
control group at T2 (the end of 72 h of reperfusion
following ischaemia) compared with the levels at T0 and
T1 (P<0.001). The BMS group behaved in a similar
manner to the saline group (Figure 3A). In contrast, CK-
MB levels in the GHRP6 group were not significantly
different when the data at T0 and T1 were compared. At
T2, a lower increase (2-fold) was detected compared with
T0 and T1 (P<0.01). Comparisons of the results from
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GHRP6 reduces myocardial ischaemic necrosis
247
Table 4
groups
Values are means+−S.D. Lethally damaged nuclei were those showing
karyorrhexis-, karyolysis- and/or karyopyknosis-typical morphology, averaged from
serial sections in 10–15 microscopic fields. Peninsulas of necrotic fibres were also
quantified. Tissue analysis was done on full-thickness left ventricular wall punch
biopsies collected from normally appearing areas adjacent to the necrotic core.
∗∗∗P <0.001 compared with the saline control group, as determined by an
unpaired Student’s t test.
Myocardial microscopic assessment in the study
Group
Parameter BMSSalineGHRP6
Necrotic fibre
peninsulas (n)
Nuclei with lethal
changes (n)
23.36+−6.11
95.33+−18.22
25.8+−4.27
90.07+−21.8
6.18+−2.22∗∗∗
27.39+−11.6∗∗∗
Figure 3
(B) levels
(A) Serum CK-MB levels were determined as a marker of myocardial fibre damage.
Acute elevation of CK-MB was detected at T1 (∗∗P <0.01) compared with T0
in the saline group. A similar observation was found with the BMS group. A
progressive increase was detected in saline and BMS groups in T2 compared
with T1 and T0 (∗∗∗P <0.001). However, no increase in CK-MB levels were
detected at T1 in the GHRP6 group (P =1.00) compared with T0. At T2, a
significant increase was detected in GHRP6-treated animals compared with T1 and
T0 (∗∗P <0.01). This increase at T2 was significantly lower (∗∗∗P <0.001)
than that observed in the control group. All statistical analyses were performed
using an unpaired Student’s t test. (B) CRP levels were not increased at T1 in any
of the groups. In contrast, at T2 this marker appeared significantly higher in
the saline and BMS groups compared with that at T0 and T1 (∗∗∗P <0.001).
In the GHRP6 group, CRP values remained similar at T2 compared with those
at T1 and T0 (all P >0.05). A significant difference at T2 compared with the
saline control group was observed (∗∗∗P <0.001). All statistical analyses were
performed using an unpaired Student’s t test.
Effect of GHRP6 on circulating CK-MB (A) and CRP
Figure 4
myocardial IGF-I
Constitutive myocardial IGF-I mRNA expression was determined from samples from
intact healthy hearts, and hearts from the saline-treated and GHRP6-treated
groups. Upper panel, a representative agarose gel of RT-PCR-amplified IGF-I and
β-actin. Lower panel, the amount of mRNA quantified by densitometry and ex-
pressed relative to β-actin.∗∗P <0.01 compared with the intact group, as
determined by an unpaired Student’s t test. No significant difference was observed
between the saline and GHRP6 groups (P =0.5476).
Effect of GHRP6 on transcriptional expression of
the study groups at the T2 sampling point indicated that
GHRP6 treatment truncated the increase in CK-MB by
approx. 70% (P<0.001).
Circulating CRP levels remained stable immediately
at T1 compared with those at T0 in all of the groups
(Figure 3B). The highest CRP levels were detected at T2
compared with those at T0 and T1 in the saline and BMS
groups (P<0.001). In contrast, no significant difference
was observed in CRP values at T2 compared with those
at T0 and T1 in GHRP6-treated animals (Figure 3B).
Myocardial expression of IGF-I
Five individual RT-PCR products from each experi-
mental group are shown in Figure 4. A significant dif-
ference in constitutive myocardial IGF-I expression
levels was observed when samples from intact healthy
hearts were compared with those receiving the ischaemic
insult (saline and GHRP6 groups; P<0.01). No differ-
ences were noted, however, when the saline and GHRP6
groups were compared.
Assessment of myocardial redox status
Assessment of some critical redox parameters within the
myocardial areas adjacent to the necrotic core provided
important insights into the effect of GHRP6 treatment
(Table 5). Intramyocardial MDA levels were elevated
over 14-fold in the saline group compared with intact
healthy hearts, whereas this increase was only 2-fold
in the GHRP6 group. Correspondingly, a similar effect
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J. Berlanga and others
Table 5
Values are means+−S.D. Statistically significant differences (P <0.05) were obtained between the groups for each parameter, as
determined by one-way ANOVA and Student–Newman–Keuls test.
Myocardial redox assessment in the study groups
Group
Parameter IntactSalineGHRP6
MDS (nmol/mg of protein)
THP (µmol/mg of protein)
SODt (units·mg−1of protein·min−1)
Catalase activity (units·mg−1of protein·min−1)
0.054+−0.007
31.78+−0.45
19081+−249
11.34+−0.26
0.766+−0.068
295.61+−8.47
9033+−229
374.86+−10.53
0.1207+−0.013
56.27+−1.01
12195+−706
34.11+−0.55
was observed for intramyocardial THP levels. A 9-fold
increase was observed in the saline group compared
with the values in intact healthy hearts, and only a 5-
fold elevation was detected in the GHRP6 group. A
substantial increase in catalase activity was observed in
thesalinegroupcomparedwiththeintacthearts.GHRP6
blocked the 10-fold increase in catalase activity. A 53%
decrease in total SODt activity was demonstrated in
the hearts from the saline group compared with the
healthy hearts. With GHRP6, the decrease in SODt
activity was only 36%.
DISCUSSION
Althoughoneofthelimitationsofthepresentstudyisthe
lack of characterization of ventricular function upon
theinductionoftheinfarctionandduringthereperfusion
phase, the fact that the treatment with GHRP6 was able
to rescue myocardial mass by more than 70% is a re-
markable finding. The present study has the merit of
being performed following a careful pre-surgical clinical
examination of the animals to ensure that an equal
allocation of healthy animals was obtained in each study
arm. Although previous studies on the cardioprotective
effects of GHRP6 in different experimental cardiopathy
settings [4–10] are confirmed and extended in the pre-
sent study, our study appears to provide the first
direct evidence of a reduction in myocardial necrosis by
GHRP6administrationinamodelofacutearterialocclu-
sion in otherwise healthy animals.
Morethan50%oftheGHRP6-treatedanimalshadno
macroscopic evidence of epicardial necrosis. In these pigs
(five out of nine), a negligible well-delimited damaged
territory (basically haemorrhagic) was detected in the
mesocardium upon dissection of the ventricle. Coinci-
dently, no pathological Q waves were found in any of
the 12 ECG leads in the pre-autopsy recordings in these
animals. The convergence of morphological, electro-
physiological and biochemical findings in the present
study emphasizes the capability of the hexapeptide to
rescue myocardial cells during I/R and its ensuing
pathological cascade.
As stated above, the effect of a multi-dose GHRP6
regimen was examined in the present study and was
intentionally planned to study the impact on specific
ventricular electrical disturbances derived from an I/R
episode. Although this dosing schedule meant that it was
difficulttodefinethecontributionofeachinterventionto
the overall clinical effect of infarct reduction, it did allow
us to conclude that GHRP6 did not ameliorate ECG-
registered rhythmic and conductance failures linked
to I/R that eventually accounted for mortality during
the surgical period (ventricular fibrillation during the
ischaemic phase). This observation is in agreement with
previous findings [10].
ThetimecourseofchangesinserumCK-MBandCRP
levels were both similar. Both parameters were found to
be elevated in all of the groups at 72 h after infarction
when compared with their respective baseline and acute
reperfusion values. Although differences exist between
CK-MB and CRP in terms of the site of production
and specific clinical significance, it is noteworthy that the
increaseinbothmarkerswasmuchlowerintheGHRP6-
treated pigs at 72 h after coronary occlusion compared
with the saline controls. This appears to confirm the
ability of GHRP6 to reduce myocardial damage, even
when the analytical systems used is intended for human,
rather than porcine, samples.
The mechanism whereby GHRP6 enhances tissue
protection during ischaemia remains unclear to date. Pre-
vious findings from our group [11] identified for the first
time that GHRP6 possessed extracardiac multi-organ
protection capabilities against prolonged ischaemia when
administered as a single prophylactic bolus. Indeed, these
effects appeared to be conferred by a putative control of
cellularoxidativestressanditsensuingpro-inflammatory
status [11]. Following this line of evidence and taking
into account that ischaemic myocardial damage is, at
least in part, associated with the local generation of
ROS(reactiveoxygenspecies)[17,18],componentsofthe
myocardial redox status were studied in macroscopically
healthy fragments obtained close to the infarct core. A
positive oxidative stress balance was confirmed in the
GHRP6 group as judged by the following findings: (i) a
significantreductionintheintramyocardialaccumulation
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GHRP6 reduces myocardial ischaemic necrosis
249
of THP and MDA, indicating that GHRP6 attenuated
ROS accumulation; and (ii) a preservation of basal
antioxidant enzyme defence levels (catalase and SODt)
otherwise altered by ROS generation [19,20]. These
positive findings may justify the contribution of GHRP6
in rescuing myocardial mass [21]. Other mechanisms for
the cardioprotective action of GHRP6 during the I/R
episode include (i) modification of myocardial regional
blood flow; (ii) acute establishment of collateral micro-
circulatory circuits near the AAR; and/or (iii) improve-
ment in general haemodynamic performance. These have
been examined previously in a similar experimental sett-
ing with negative results [10].
GHstimulatesmyocardialIGF-Isynthesis,whichloc-
ally promotes cardiomyocytes division, differentiation
and survival [22]. Thus myocardial IGF-I expression
level was examined as a candidate effector distal to
peptide-mediated GH release [23]. RT-PCR experiments
indicated that, in the groups receiving ischaemia (saline
and GHRP6 groups), higher IGF-I expression was
observed compared with levels detected in intact hearts.
This possibly indicates a hypoxia-associated response or,
alternatively, an ongoing local repair process. Under the
sampling schedule used in the present study, GHRP6 did
not amplify myocardial IGF-I expression. This observ-
ation appears to substantiate in vivo [10] and in vitro
[24,25] observations that show that GHRP6 and other
peptidyl GH secretagogues may have direct cardio-
protective effects independent of the GH/IGF-I system.
However, this does not exclude GHRP6-mediated GH
released in a pulsatile fashion having beneficial cardiac
effects.
Activation of ERK1/2 (extracellular-signal-regualted
kinase 1/2) and PI3K (phosphoinositide 3-kinase)/Akt
pathways by ghrelin and other cytoprotective agents
play a critical role in preventing cells death under
different stressful conditions [24]. Cell survival against
pro-oxidant cytotoxicity also requires the involvement
of PI3K/Akt [21]. Other hypothetical mechanisms for
GHRP6-mediated cardiomyocytes protection include
an improvement in myocardial metabolic efficiency by
activation of glycolysis and reduction in reperfusion-
induced hypermetabolism. Previous studies have shown
thatcellsurvivalviaAktactivationislargelydependenton
glycolytic metabolism and energy optimization [26,27].
In summary, GHRP6 treatment substantially reduced
myocardial infarct size following an acute and sudden
ischaemic event. The replication of this finding in the
clinical arena may improve the prognosis of individuals
affected by myocardial infarction.
ACKNOWLEDGMENTS
We thank Dr Juan Valiente from the National Institute
of Cardiology, Havana, Cuba for his kind assistance in
reviewing the manuscript prior to submission, and to
Dr Antonio Enamorado, Director General of the
HermanosAmeijeirasHospital,forthesupportprovided
duringthestudy.ThestudywasfundedbytheBiomedical
ResearchDirectionoftheCenterforGeneticEngineering
and Biotechnology, Havana, Cuba.
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Received 4 May 2006/7 September 2006; accepted 21 September 2006
Published as Immediate Publication 21 September 2006, doi:10.1042/CS20060103
C ?2007 The Biochemical Society