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Am J Physiol Endocrinol Metab 292: E1418 E1425, 2007.

First published January 23, 2007; doi:10.1152/ajpendo.00335.2006.

Growth hormone receptor deficiency in mice results in reduced systolic blood


pressure and plasma renin, increased aortic eNOS expression, and altered
cardiovascular structure and function
E. Egecioglu,1 I. J. Andersson,1 E. Bollano,2,3 V. Palsdottir,4 B. G. Gabrielsson,4 J. J. Kopchick,5
O. Skott,6 P. Bie,6 J. Isgaard,7 M. Bohlooly-Y,8 G. Bergstrom,1,7 and A. Wickman1
Departments of 1Physiology and 2Cardiology, 3Wallenberg Laboratory for Cardiovascular Research, 4Department of
Metabolism and Cardiovascular Research, Sahlgrenska Academy, Goteborg University, Gothenberg, Sweden; 5Edison
Biotechnology Institute and Department of Biomedical Sciences, Ohio University, Athens, Ohio; 6Department of Physiology
and Pharmacology, Institute of Medical Biology, Syddansk University, Odense, Denmark; 7Department of Clinical
Physiology, Sahlgrenska Academy, Goteborg University, Gothenberg, Sweden; and 8AstraZeneca R&D, Molndal, Sweden
Submitted 11 July 2006; accepted in final form 12 January 2007

Egecioglu E, Andersson IJ, Bollano E, Palsdottir V, Gabrielsson


BG, Kopchick JJ, Skott O, Bie P, Isgaard J, Bohlooly-Y M, Bergstrom G, Wickman A. Growth hormone receptor deficiency in mice
results in reduced systolic blood pressure and plasma renin, increased
aortic eNOS expression, and altered cardiovascular structure and function. Am J Physiol Endocrinol Metab 292: E1418 E1425, 2007. First
published January 23, 2007; doi:10.1152/ajpendo.00335.2006.To
study the role of the growth hormone receptor (GHR) in the development of cardiovascular structure and function, female GHR genedisrupted or knockout (KO) and wild-type (WT) mice at age 18 wk
were used. GHR KO mice had lower plasma renin levels (12 2 vs.
20 4 mGU/ml, P 0.05) and increased aortic endothelial NO
synthase (eNOS) expression (146%, P 0.05) accompanied by a
25% reduction in systolic blood pressure (BP, 110 4 vs. 147 3
mmHg, P 0.001) compared with WT mice. Aldosterone levels were
unchanged, whereas the plasma potassium concentration was elevated
by 14% (P 0.05) in GHR KO. Relative left ventricular weight was
14% lower in GHR KO mice (P 0.05), and cardiac dimensions as
analyzed by echocardiography were similarly reduced. Myograph
studies revealed a reduced maximum contractile response in the aorta
to norepinephrine (NE) and K (P 0.05), and aorta media thickness
was decreased in GHR KO (P 0.05). However, contractile force
was normal in mesenteric arteries, whereas sensitivity to NE was
increased (P 0.05). Maximal acetylcholine-mediated dilatation was
similar in WT and GHR KO mice, whereas the aorta of GHR KO mice
showed an increased sensitivity to acetylcholine (P 0.05). In
conclusion, loss of GHR leads to low BP and decreased levels of renin
in plasma as well as increase in aortic eNOS expression. Furthermore,
GHR deficiency causes functional and morphological changes in both
heart and vasculature that are beyond the observed alterations in body
size. These data suggest an important role for an intact GH/IGF-I axis
in the maintenance of a normal cardiovascular system.
cardiac structure; cardiac function; vascular structure; vascular function; endothelial nitric oxide synthase; growth hormone receptor-null
mice

structural and functional changes of the cardiovascular system


are, however, unclear.
GH-deficient patients have a higher prevalence of cardiovascular morbidity and mortality than the general population
(16, 20, 26). However, patients with GH insensitivity/resistance (Laron syndrome), which is a much rarer condition, are
less well studied in relation to cardiovascular mortality and
morbidity. These patients have reduced cardiac dimension,
with reductions in left ventricular (LV) volume and mass, as
well as in stroke volume and cardiac output (CO) (11). However, systolic and diastolic functions appear intact, and the
cardiac response to exercise is normal. Thus, it appears that the
heart, despite its smaller size, can meet the metabolic need in
GH-resistant patients. To our knowledge, consequences of GH
insensitivity on vascular function and structure have not been
studied in patients with GH insensitivity/resistance, whereas
patients with GH deficiency, conversely, display endothelial
dysfunction (10) and decreased systemic formation of nitric
oxide (NO) (1).
An animal model resembling GH resistance is the GH
receptor (GHR)/GH-binding protein gene disrupted, or knockout (GHR KO), mouse (33). Although these mice secrete GH,
there are no biological effects because of the lack of the GHR.
It has previously been reported that the resultant mouse phenotype has very low levels of serum insulin-like growth factor
I (IGF-I) and insulin, loss of GHR and GH-binding protein, and
an 50% weight reduction compared with littermates at 12 wk
of age (9, 33).
The aim of the study was to investigate the importance of an
intact GH/IGF-I axis in the development of normal cardiovascular function and morphology, in particular, the importance of
the GHR in cardiac and vascular function and structure.
MATERIALS AND METHODS

(GH) activity,
such as GH deficiency or GH insensitivity/resistance, or GH
excess as exemplified in acromegaly, are associated with an
increase in cardiovascular disease (5, 26). The mechanisms by
which abnormally low or high levels of GH signaling cause

Animals. The GHR KO mouse was generated as previously described (33). Homozygous GHR KO and wild-type (WT) littermate
females (Sv129Ola-Balb/c background) were identified by PCR analysis of DNA from tail biopsy specimens as previously described (33).
Animals were housed together with littermate controls during the
whole study. The environment of the animal rooms was controlled

Address for reprint requests and other correspondence: E. Egecioglu, Dept.


of Physiology, The Sahlgrenska Academy, Goteborg University, PO Box 434,
SE-405 30 Gothenberg, Sweden (e-mail: emil.egecioglu@medic.gu.se).

The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

CONDITIONS LINKED TO REDUCED GROWTH HORMONE

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0193-1849/07 $8.00 Copyright 2007 the American Physiological Society

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GH RECEPTOR AND CARDIOVASCULAR STRUCTURE AND FUNCTION

with a 12:12-h light-dark cycle, a relative humidity of 4555%, and a


temperature of 20C. The mice had free access to tap water and
standard pellet chow. The ethics committee of Goteborg University
gave prior approval of the animal procedures. Eighteen-week-old
mice were used in the study.
Systolic blood pressure. Systolic blood pressure (SBP) was measured using a computerized noninvasive tail-cuff system (RTBP
Monitor; Harvard Apparatus, South Natick, MA). Conscious animals
(n 8 in each group) were kept in a restraining cylinder with a
standard acclimatization time of 10 min, and the tail was gently heated
before every recording session. The recordings were performed in the
afternoon on four consecutive days with at least 10 measurements at
each recording. Final SBP was obtained from the three last days of
recording by averaging the mean values.
Echocardiography. Mice (GHR KO, n 8; WT, n 7) were
anesthetized with isoflurane (Baxter Healthcare, Chicago, IL). The
chest was shaved and a peeling cream applied, removing remaining
hair. Electrocardiography (ECG) leads were placed on the extremities;
a warming pad was used to maintain body temperature. Cardiac
ultrasound studies were performed using a commercially available
ultrasonograph (ATL HDI 5000 SonoCT; Phillips Ultrasound,
Bothell, Seattle, WA). Briefly, a 15-MHz linear transducer was used
to obtain two-dimensional parasternal short-axis images close to the
papillary muscle. This served as a guide for M-mode tracing. twodimensional guided, pulsed, Doppler was used to record the estimated
peak LV outflow tract velocity and the mitral inflow velocities (E and
A wave and their ratio). This was done using minimal sample size and
a pulsed frequency of 5 MHz. All tracings were recorded at a sweep
of 100 mm/s and were stored on magnetic optical discs for offline
measurements. Offline measurements were done by one observer
blinded to the type of mice.
M-mode measurements of LV internal diameters and wall thickness in diastole and systole were made using the convention of the
American Society of Echocardiography (www.asecho.org). End diastole was taken at the onset of the QRS complex, and end systole was
taken at the peak inward of interventricular septum motion. Four or
more beats were averaged for each measurement.
LV fractional shortening (FS) was calculated as follows: (LVIDdLVIDs)/LVIDd 100, where LVIDd and LVIDs are LV internal
diameters in diastole (d) and systole (s), respectively. Relative wall
thickness and velocity of circumferential shortening (Vcf) were calculated using formulas described previously (30).
ECG. The mice were anesthetized with isoflurane and ECG leads
were placed on the extremities (GHR KO n 7, WT n 7). A
heating pad was used to maintain body temperature. ECG was
recorded at 2 kHz using Pharmlab 3.0 (AstraZeneca, Molndal,
Sweden) for a minimum of 20 min, and the level of anesthesia was
adjusted to keep heart rate stable at 400 beats/min. Time parameters
were measured using Pharmlab 3.0, and a mean QRS complex was
generated as a mean of 20 beats. A graphic interface of the
analysis program allowed visual reviewing and manual editing of
the detected events. PQ time was defined as the time from start of the
P wave to the start of the Q or R wave, the QRS time as the time from
the start of the Q or R wave to the neutralization of the R wave, and
the QT time as the start of the Q wave or R wave to the neutralization
of the T wave. QT time was also correlated to the RR interval due to
heart rate influence on the QT time. RR interval and R amplitude were
also calculated.
Plasma renin, aldosterone, sodium, and potassium measurements.
At 17 wk of age, plasma renin concentration was measured (n 17
in each group). Following a very short anesthesia (isoflurane) blood
was taken from the orbital plexa, and the plasma was frozen for later
measurement of plasma renin concentration by radioimmunoassay of
angiotensin I by use of the antibody-trapping technique (19). Only
results with linearity in serial dilutions (between 50- and 1,000-fold)
were accepted. Renin values were standardized with renin standards
obtained from the National Institutes for Biological Standards and
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Control (Potters Bar, Hertfordshire, UK) and are expressed in milliGoldblatt units per milliliter (mGU/ml).
Plasma aldosterone was determined using a commercial kit (n
12 in each group, COAT-A-COUNT; Diagnostic Products, Los
Angeles, CA).
Sodium and potassium concentrations in plasma were determined
using flame photometry (n 12 in each group, model ILS 943;
Instrumentation Laboratory, Lexington, MA).
Ex vivo vascular function. GHR KO (n 8) and WT (n 8) mice
were weighed and anesthetized with pentobarbital sodium (60 mg/kg,
Apoteksbolaget, Gothenberg, Sweden) before the aorta, mesenteric
arteries, and heart were dissected out. The vessels were placed into
physiological salt solution (PSS). The aorta was separated into three
parts; one part was fixed in formalin for further morphological
analysis, one part was used for ex vivo functional tests, and the third
part was trimmed free of surrounding tissues and frozen in liquid
nitrogen and saved for further mRNA analysis. The heart was separated into LV including septum and right ventricle (RV) and weighed.
Ring segments of thoracic aorta (3 mm, GHR KO n 8, WT n
8) and mesenteric arteries (GHR KO n 10, WT n 13) were
mounted in an organ bath (Department of Physiology, Goteborg
University, Gothenberg, Sweden) and a Multi myograph 610M (Danish Myo Technology, Aarhus, Denmark), respectively, as described
before (15). Aortic rings were stretched to 3 mN and equilibrated for
30 min. Before the pharmacological protocol, they were further
stretched to 12 mN and allowed to stabilize. The internal circumferences of the mesenteric arteries (GHR KO n 10, WT n 13) were
normalized according to the manufacturers protocol. All vessels were
activated with norepinephrine (NE, 105 mol/l) and KCl (K, 101
mol/l) with 20-min washout between each procedure. The cumulative
concentration-response relationship to NE was then studied (aorta
109 to 105 mol/l, mesenteric arteries 108 to 105 mol/l). The
endothelium-dependent relaxation response was studied by cumulative concentration response to acetylcholine (ACh, 109 to 105
mol/l) on vessels precontracted to 50 100% of maximal recorded NE
contraction. A second concentration-response to ACh was performed
in the presence of the NO synthase (NOS) inhibitor N-nitro-Larginine (L-NNA, 104 mol/l). Sodium nitroprusside (Sigma, St.
Louis, MO; 105 and 104 mol/l) was applied after each ACh
concentration-response to validate the endothelium-independent relaxation of the smooth muscle cells.
Morphological aorta analysis. Aortic sections (n 6 in each
group) were paraffin imbedded, sectioned in a microtome, and
mounted on slides for hematoxylin staining. Aortic wall thickness and
number of muscle layers were measured on these sections. Wall
thickness was measured and muscle layers were counted by a blinded
observer on five different sections of the aortic wall at 40 magnification.
Real-time PCR analysis. Total RNA from aorta (n 8 in each
group) was extracted with Tri Reagent (Sigma), and concentrations
were determined by Ribo Green (Molecular Probes, Leiden, The
Netherlands) following the manufacturers protocol. Standard cDNA
from pooled aortic RNA was synthesized in parallel with the sample
cDNAs. A standard curve, obtained by serial dilution of the standard
cDNA (range 0.6 to 80 ng original RNA per well), was included on
each plate.
PCR primers for GAPDH, caveolin-1, and extracellular superoxide
dismutase (ecSOD) were designed using LightCycler Probe Design
Software (version 1.0; Roche Diagnostics, Mannheim, Germany).
Primer sequences and thermal conditions for each gene are listed in
Table 1. Relative quantification of caveolin-1 and ecSOD mRNA
expression was performed using a LightCycler (Roche Diagnostics)
and FastStart Master SYBR Green I (Roche Diagnostics), and expression levels were normalized to GAPDH. Transcript levels of eNOS,
IGF-I, and prolactin receptor were analyzed using predesigned TaqMan assay-on-demands (Applied Biosystems, Foster City, CA) and
performed on an ABI Prism 7900HT Sequence Detection System

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GH RECEPTOR AND CARDIOVASCULAR STRUCTURE AND FUNCTION

Table 1. Primer sequences, times, and temperatures for denaturizing, annealing, and elongation for the genes GAPDH,
PPIA, caveolin-1, and ecSOD used in real-time quantitative PCR
Gene

Denaturation

Annealing

Elongation

Oligonucleotide

GAPDH

0 s, 95C

4 s, 67C

3 s, 72C

Forward primer
Reverse primer
Forward primer
Reverse primer
Forward primer
Reverse primer
Forward primer
Reverse primer

PPIA

15 s, 95C

60 s, 60C

Caveolin-1

15 s, 95C

4 s, 58C

8 s, 72C

ecSOD

15 s, 95C

4 s, 56C

9 s, 72C

Sequence

5-GTC
5-TGC
5-AGG
5-TGC
5-GAC
5-AGG
5-AGC
5-GTC

GTG
CTG
GTT
CAT
GAC
AAG
TAG
CCC

GAT
CTT
CCT
TAT
GTG
GAG
GAC
GAA

CTG
CAC
CCT
GGC
GTC
AGA
GAC
CTC

ACG
CAC
TTC
GTG
AAG
ATG
GAA
ATG

TGC C-3
CTT CT
ACA GAA TT-3
TAA AGT C-3
AT-3
GC-3
G-3
C-3

GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PPIA, cyclophilin A; ecSOD, extracellular superoxide dismutase.

(Applied Biosystems). Reagents (TaqMan Universal PCR Mastermix,


Applied Biosystems) and reaction conditions were used according to
the manufacturers instructions. Cyclophilin A (PPIA; CyberGene,
Huddinge, Sweden; Table 1) was used as a reference gene for eNOS
and prolactin receptor and -actin for IGF-I. All standards and
samples were analyzed in triplicate.
Statistics. Values are expressed as means SE. If not stated
otherwise, two-tailed t-tests for unpaired data were used for statistical
comparisons between groups. Variables without homogeneity in variance or normal distribution were analyzed with a Mann-Whitney
U-test. P values 0.05 were considered to be statistically significant.
RESULTS

Systolic blood pressure was reduced by 25% in GHR KO


mice compared with WT (KO 110 4 vs. WT 147 3
mmHg, P 0.001; Fig. 1). Since the tail cuff technique is
sensitive to the relation between the cuff and the size of the tail,
we apprehended that the smaller tail of the GHR KO animals
possibly could affect our measurements. However, when two
different sizes of WT mice on a different background (C57/
BL6) were studied (18 vs. 28 g), similar systolic blood pressures were recorded in both groups [18 g, n 5: 112 2 vs.
28 g, n 5: 110 2 g, not significant (NS); unpublished
observations].

Fig. 1. Systolic blood pressure was reduced by 25% in growth hormone


receptor (GHR) knockout (KO) vs. wild-type (WT) mice (*P 0.05). mGU,
milliGoldblatt units.
AJP-Endocrinol Metab VOL

The plasma renin concentrations were 40% lower in GHR


KO compared with WT mice (KO 12 2 vs. WT 20 4
mGU/ml, P 0.05 as analyzed with Mann-Whitney U-test;
Fig. 2A). No change in aldosterone levels was found in GHR
KO compared with WT mice (KO 815 101 vs. WT 812
46 pg/ml, NS; Fig. 2B).
The plasma potassium concentrations were increased by
14% in GHR KO compared with WT mice (KO 7.41 0.28
vs. WT 6.51 0.27 mmol/l, P 0.05 as analyzed with
Students t-test; Fig. 2C). Sodium concentrations were unchanged in GHR KO (KO 157 2 vs. WT 155 2 mmol/l;
Fig. 2D).
The levels of aortic eNOS mRNA were increased by 146%
in GHR KO compared with WT (KO 16.5 3.7 vs. WT 6.7
0.9, P 0.05; Fig. 3).
GHR KO mice had 53% decreased body weight compared
with WT (KO 17 1 vs. WT 36 3 g, P 0.001; Table 2).
The relative LV weights were also lower in the GHR KO mice
compared with WT (KO 22.7 1.6 vs. WT 26.5 0.9 mg/10
g body wt, P 0.05). There was no difference in the relative
RV weight between GHR KO and WT mice (KO 5.9 0.3 vs.
WT 7.0 0.6 mg/10 g body wt).
In GHR KO mice, posterior wall thickness was decreased by
25% (P 0.05) and LVDd was similarly decreased by 28%
(P 0.05) compared with WT (Table 2). The resultant relative
wall thickness was similar in both groups. When %FS and Vcf
were calculated, GHR KO mice displayed decreased %FS and
Vcf by 11 and 24%, respectively (P 0.05; Table 2), suggesting decreased systolic cardiac function. The diastolic cardiac function was also studied by calculating the E/A quote,
but no differences were detected between the groups. CO was
decreased by 48% (P 0.05; Fig. 4A) in GHR KO; but when
CO was related to body weight, no differences between the
groups were detected (Table 2 and Fig. 4B).
During ECG measurements, heart rate was kept at 400
beats/min in both groups (KO 412 17 vs. WT 448 8
beats/min). GHR KO mice showed longer QT time compared
with WT (KO 27.7 1.0 vs. WT 22.7 1.8 ms). However,
when the QT time was corrected for the small difference in
heart rate (QTc), no significant difference was detected between the groups (KO 2.26 0.05 vs. WT 1.95 0.06, P
0.08). PQ time and QRS time were similar in both groups (PQ:
KO 37.5 0.3 vs. WT 35.7 1.0; QRS: KO 12.1 0.6 vs.
WT 10.9 0.4 ms).
In the mesenteric arteries, no difference in maximum contractile response to K and NE could be detected between

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Fig. 3. GHR KO mice had increased mRNA expression of endothelial NO


synthase (eNOS) normalized to cyclophilin A (PPIA) in the aorta (*P 0.05).

Fig. 2. A: plasma renin concentrations was decreased by 40% in GHR KO vs.


WT (*P 0.05, Mann-Whitney U-test; n 17 in each group). No difference
in aldosterone (B) or sodium (C) was found between GHR KO and WT
animals. D: potassium levels in plasma were increased by 14% in the GHR KO
vs. WT mice (*P 0.05, Students t-test; n 12 in each group).

AJP-Endocrinol Metab VOL

GHR KO and WT mice (K: KO 1.73 0.09 vs. WT 1.91


0.068 nM/mm, respectively; NE: KO 1.59 0.11 vs. WT
1.58 0.1 nM/mm, respectively; Fig. 5A). GHR KO showed
an increased sensitivity to NE compared with WT in the
mesenteric arteries (log EC50: KO 5.5 0.1 vs. WT 5.2
0.1 log mol/l, respectively, P 0.05; Fig. 5C). Sodium
nitroprusside was used to test the function of the smooth
muscle cells and caused a less profound relaxation in the
mesenteric arteries in GHR KO compared with WT (KO
72.0 3.9 vs. WT 83.4 2.6%, respectively, P 0.05). In
the mesenteric arteries, there was no difference in the maximal
relaxation response induced by ACh relative to the maximal
endothelium-independent relaxation induced by sodium nitroprusside (KO 49.4 6.1 vs. WT 40.9 6.7%) and no
difference in the sensitivity to ACh (KO 6.6 0.058 vs. WT
6.9 0.18; Fig. 5E). Blockade of NOS by L-NNA reduced
ACh-induced dilations significantly more in the mesenteric
arteries in GHR KO compared with WT (KO 11.2 5.2 vs.
WT 27.5 4.1%, respectively, P 0.05; Fig. 5E).
In the aorta, the maximum contractile response to K and
NE was reduced in GHR KO compared with WT (K KO:
0.7 0.1 vs. WT 1.4 0.1 mN/mm, respectively; NE: KO
1.2 0.07 vs. WT 2.3 0.1 mN/mm, respectively, P 0.05
for both; Fig. 5B). There were no differences in the sensitivity
to NE (log EC50: KO 7.35 0.11 vs. WT 7.44 0.13 log
mol/l; Fig. 5D) or in ACh-induced maximal relaxation relative
to the maximal endothelium-independent relaxation induced
by sodium nitroprusside in aorta (KO 54.2 6.6 vs. WT
44.5 6.7%: Fig. 5F). However, sensitivity to ACh was
increased in aorta in GHR KO compared with WT (log EC50:
KO 7.6 0.1 vs. WT 7.1 0.2 log mol/l, respectively,
P 0.05). The presence of L-NNA totally abolished AChinduced relaxations (Fig. 5F), and sodium nitroprusside caused
profound relaxation in aorta in both GHR KO and WT (KO
98.5 1.5 vs. WT 100 1.9%, respectively).
The thickness of the GHR KO aortic media was significantly
reduced by 29% compared with WT (KO 45.83 4.5 vs. WT
64.58 4.78 m, P 0.05), whereas no changes in the

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GH RECEPTOR AND CARDIOVASCULAR STRUCTURE AND FUNCTION

Table 2. Body weight and various variables from ECG measurements


n

WT
GHR KO
P value

7
8

BW,
g

HR,
beats/min

LVDd,
mm/10 g BW

LVDs,
mm/10 g BW

PWTh,
mm/10 g BW

RWTh

Vcf,
circ/s

FS,
%

E Wave,
cm/s

A Wave,
cm/s

E/A

363
171
0.001

40455
33231
NS

0.980.04
1.470.11
0.001

0.380.02
0.670.05
0.001

0.280.01
0.440.01
0.001

0.570.02
0.620.05
NS

10.030.97
7.630.57
0.04

612
552
0.02

0.600.05
0.510.03
NS

0.390.09
0.300.04
NS

1.750.26
1.800.14
NS

WT, wild type; GHR KO, growth hormone receptor knockout; BW, body weight; HR, heart rate; LVDd, left ventricular diameter (diastole); LVDs, left
ventricular diameter (systole); PWTh, posterior wall thickness; RWTh, relative wall thickness; Vcf, velocity of circumfential shortening; FS, fractional
shortening; NS, not significant.

number of muscle layers could be found between GHR KO and


WT (average: KO 4.4 vs. WT 4.6, respectively).
The levels of aortic caveolin-1 mRNA tended to be lower in
the GHR KO animals (KO 0.6 0.08 vs. WT 0.89 0.15,
P 0.1). Expression levels of ecSOD (KO 1.55 0.52 vs.
WT 1.43 0.70, NS) and IGF-I mRNA (KO 1.43 0.50 vs.
WT 0.73 0.15, NS) were unchanged as was that of the
prolactin receptor (KO 217.12 122.8 vs. WT 270.64
128.6, NS). However, variances in the mRNA levels tended to

Fig. 4. A: cardiac output (CO) was reduced in GHR KO vs. WT mice (*P
0.05). B: when CO was normalized to body weight, there was no difference
between the groups.
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be high in some of the measurements. Therefore, lack of


significance in the statistical analysis of differences between
groups does not necessarily indicate that there was no biological difference.
DISCUSSION

In the present study, we have shown that lack of GHR


signaling causes a reduction in systolic blood pressure and
plasma renin levels as well as an increase in aortic eNOS
expression. Furthermore, cardiac weight and volume were
reduced to a larger extent than could be explained by the lower
body weight of the GHR KO mouse. However, despite a
reduced global systolic function, the GHR KO mice seem to be
able to produce a CO that matches their reduction in body
weight and metabolic rate but display a lower systolic blood
pressure. The aorta of the GHR KO shows a markedly hypotrophic phenotype with a reduction in media thickness and a
reduced maximal contractile response. Sensitivity to AChmediated dilatation is increased in the aorta with a matching
increase in eNOS mRNA expression. The resistance vasculature appears normally developed but with an alteration in the
factors responsible for ACh-mediated dilatation. Thus, we
conclude that functional GH signaling is important for the
maintenance of normal cardiac and large vessel structure and
function.
GHR KO mice had reduced systolic blood pressure, which
also has been found in hypophysectomized (Hx) rats (13, 24,
31). The mechanisms underlying this reduction remain unclear,
but it has been speculated that a generally hypotrophic cardiovascular system could be causative (12). The present study
offers several explanations for the reduction in systolic blood
pressure seen in GHR KO mice. The observed reduction in
systolic cardiac function may reduce systolic blood pressure.
This effect could further be augmented by the altered endothelial function toward a more relaxed state with increased eNOS
expression and increased NO release, resulting in a reduction
in total peripheral resistance. Furthermore, reduced large-vessel compliance could be underlying the cardiovascular changes
such that less contractile force would be needed for peak
ejection, resulting in reduced systolic pressure. Another important finding is the significant reduction in plasma renin activity.
A general response to a reduction in blood pressure would be
to increase plasma renin levels (14). Thus, a possible altered
regulation of plasma renin levels may be of importance for the
lower blood pressure in the KO animals. However, the reduction in plasma renin was not accompanied by a concomitant
reduction in plasma aldosterone. This, in turn, might possibly
be explained by the slightly increased plasma potassium levels
in the GHR KO. Speculatively, the elevated potassium levels

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Fig. 5. Maximal contraction in response to norepinephrine (NE) was unchanged in mesenteric arteries (A) and decreased in the aorta of GHR KO compared with
WT mice (*P 0.05; B). Relative response to NE, expressed as %maximal contractile force, was increased in mesenteric arteries (*P 0.05; C) but unchanged
in the aorta of GHR KO compared with WT (D). E: response to ACh induced dilatation after N-nitro-L-arginine (L-NNA) treatment was impaired in mesenteric
arteries of GHR KO (*P 0.05). SNP, sodium nitroprusside. F: however, sensitivity to ACh was increased in aorta of GHR KO vs. WT mice (*P 0.05).

may contribute to the maintained aldosterone levels. Systolic


and diastolic blood pressure are essentially independent of
mammal size (8), diminishing the significance of size differences between the GHR KO and WT. However, even though
we had a reasonable number of observations in the aldosterone
(n 12) measurements, the lack of statistical significance
between the groups does not exclude a potential biological
difference.
ECG and posteuthanasia weights revealed a reduction in
cardiac volume and mass that exceeded the observed reduction
AJP-Endocrinol Metab VOL

in body weight in GHR KO mice. This is in agreement with


data from Hx rats, which display reductions in relative LV
weight (31) and cardiac dimensions, estimated by ECG (Bollano E, personal communication).
Global systolic function, measured as ejection fraction or
fractional shortening, was also reduced in GHR KO mice
compared with WT. It has previously been shown that GH
affects myocardial contractility via regulation of calcium handling during excitation/contraction coupling (4, 28). Thus, the
lack of GHR signaling in GHR KO mice may result in a

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GH RECEPTOR AND CARDIOVASCULAR STRUCTURE AND FUNCTION

defective cardiac calcium handling and resultant reduction of


myocardial contractility. Furthermore, CO was reduced in
GHR KO mice, which is in accord with the situation in Hx rats
(17, 27) as well as for patients with GH resistance and/or GH
insensitivity (11). The GHR KO mice showed a 48% decrease
in CO, which is closely related to a similar reduction in body
weight (43% in this part of the study). This may suggest
that the GHR KO mouse is small and has a small heart that,
despite systolic dysfunction, appears able to produce a resting
CO sufficient to supply the body at rest. The cardiac features of
GHR KO mice, in part, agree with those in adults with primary
GH resistance, which exhibit reduced cardiac dimensions but
normal systolic and diastolic cardiac function at rest (11). The
cardiac function of GHR KO mice is also comparable with
the situation in GH-deficient patients (22). Taken together,
these data suggest that a functional GHR signaling is of
importance for both cardiac structural development and cardiac
contractile function.
In accord with the situation in the heart, the aorta of the
GHR KO mice appeared hypotrophic, evidenced by a morphological reduction in the mean media thickness. This was in
agreement with the finding of a marked reduction in the
contractile function of the aortic segments in response to both
NE and K. Thus, as for the heart, it appears that intact GH
signaling is of importance for development of both functional
and structural aspects of large vessels.
The functional studies of GHR KO mesenteric vessels revealed a close to normal function with an intact contractile
response. However, GHR KO mice showed an increased sensitivity to NE. Effect of GH on NE sensitivity is in line with
findings from studies of Hx rats, which consistently show
increased NE sensitivity (12, 13, 24, 25) and also with results
from bovine GH transgenic mice, in which overproduction of
GH results in decreased sensitivity to NE (2). The mechanisms
for the alterations in NE sensitivity caused by GH are not
known. One of the best known causes of changes in NE
receptor sensitivity is compensatory changes in response to
alterations in sympathetic outflow. Another established fact is
that a chronic decrease in circulating catecholamines upregulates the number and/or sensitivity of adrenoceptors (6). Decreased release of catecholamines and adrenal catecholaminesynthesizing enzymes have been described after hypophysectomy (23, 32). Thus, a compensatory upregulation of the
vascular adrenoceptors due to reduced sympathetic activity
may be responsible for the increased NE sensitivity. Reduced
sympathetic activity is supported by the low blood pressure and
low plasma renin activity in GHR KO mice but would be in
contrast to the situation in GH-deficient patients, reported to
have increased sympathetic activity (29).
Endothelial function was intact in both aorta and mesenteric
artery from GHR KO mice. In fact, the sensitivity toward ACh
was increased in the aorta of GHR KO mice compared with
WT mice. L-NNA effectively blocked the ACh-mediated dilatory response in the aorta of both WT and GHR KO mice,
suggesting that it is principally mediated by NO. In contrast,
L-NNA minimally affected the dilatory response to ACh in WT
mesenteric arteries, suggesting that other factors, such as prostaglandins, endothelial-derived relaxing factor, or endothelialderived hyperpolarizing factor, are responsible for the vasodilatation in this vascular segment. Interestingly, in GHR KO
mesenteric arteries, L-NNA effectively blocked vasodilatation,
AJP-Endocrinol Metab VOL

suggesting that vasodilatation is mediated by NO. The observed change in endothelial function in the direction of a more
NO-dependent vasodilatation is hard to explain. It is in line
with our observation of increased expression of eNOS mRNA
in the aorta of GHR KO mice, but to directly link it to the loss
of GH action, further studies are needed.
In contrast to the GHR KO mice, GH-deficient patients
display impaired endothelial function (10), which may be
linked to a decreased systemic production of NO (1). It is
possible that GHR KO mice have developed compensatory
systems that maintain NO production and endothelial function.
A possible explanation to the increased eNOS expression and
NO dependency is the observation of high levels of prolactin in
GHR KO mice (18). It has been shown that prolactin activates
the NO system (3), and its receptors are located in endothelial
cells (21). Thus, increased activity of NO by prolactin might
help improve NO-dependent endothelial function in GHR KO
mice. Due to these observations, aortic prolactin receptor gene
expression in GHR KO and WT mice was measured, but the
levels did not differ between the groups.
Loss of GHR causes functional and morphological changes
in both heart and vasculature beyond the observed alterations
in body size. Despite these changes, the GHR KO mice have an
extended longevity (7). Cardiac changes in GHR KO mice
have similarities with those observed in GH-resistant and
GH-deficient patients, but important differences are observed
relating to vascular function. All together, this study suggests
an important role for an intact GH/IGF-I axis in the development of a normal cardiovascular system.
ACKNOWLEDGMENTS
We thank Jing Jia for excellent help with blood pressure measurements,
Maria Sundling for excellent help with the aortic functional tests, and
SWEGENE Centre for Mouse Physiology and Bio-Imaging.
GRANTS
This study was supported by the King Gustaf V:s and Queen Victoria
foundation (A. Wickman), the Novo Nordisk foundation (A. Wickman), the
Swedish Hypertension Society (A. Wickman), the Swedish Society of Medicine (A. Wickman), the Swedish Research Council (G. Bergstrom, 12 580),
and the Swedish National Heart and Lung Foundation (G. Bergstrom).
SWEGENE supports the postdoctoral position of A. Wickman. J. J. Kopchick
is supported, in part, by the State of Ohio Eminent Scholars Program, which
includes a gift by Milton and Lawrence Goll.
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