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INTRODUCTION |
Several observations implicate a role for growth hormone
(GH)1 in modulation of
cardiac structure and function (1). Patients with excess endogenous GH
(i.e. acromegaly) suffer from cardiac complications
including biventricular hypertrophy, impaired diastolic filling, and
decreased cardiac performance on effort due to diastolic and systolic
dysfunction (2). Patients with chronic GH deficiency also show cardiac
abnormalities; in general, the data support the presence of a
hypokinetic cardiac syndrome in patients with GH deficiency that can be
reversed with GH replacement therapy (3-5). Fazio et al.
(6) reported that GH therapy in patients with idiopathic dilated
cardiomyopathy was associated with significant improvement in left
ventricular ejection fraction, isovolumic relaxation time, and
efficiency of myocardial energy utilization. Subsequent to these
landmark findings, some studies have supported a beneficial effect of
exogenous GH on cardiac function (7), whereas other investigators were
unable to demonstrate salutary effects of GH on cardiac function in
patients with heart failure (8).
A particularly well studied animal model is that of the transplanted
GH-secreting pituitary tumor cell line, GH3. In this model
of GH excess, there is increased myocardial contractility and calcium
sensitivity of myocardial contractile proteins (1). Similarly, normal
rats given recombinant GH show an increase in left ventricular mass, as
well as an increase in several aspects of cardiac performance (9). In
the rodent model of myocardial infarction, administration of GH results
in improvements in myocardial contractility, left ventricular
end-systolic and end-diastolic pressures, and cardiac index with no
increase in the size of the infarct (10-13). In general, animal models
of GH deficiency also support a role for GH in the maintenance of
cardiac structure and function. Thus, genetically GH-deficient dwarf
mice show cardiac abnormalities that can be reversed following GH
therapy (14). However, hypophysectomized rats given GH show little
improvement in ventricular function, indicating that GH may cooperate
with other factors (e.g. thyroid hormone) in its effects on
the heart (15).
In the intact animal, GH increases the circulating levels of IGF-1 by
stimulating the production of IGF-1 (16). IGF-1 itself induces
hypertrophy and alters gene expression in isolated cardiomyocytes and
increases myocardial contractility (17, 18). IGF-1 may also serve to
inhibit cardiomyocytes from undergoing apoptosis following infarction
(19). However, to date, studies have not been able to demonstrate
direct effects of GH on isolated cardiomyocytes. Thus, Ito et
al. (20) reported that, whereas IGF-1 was able to induce
hypertrophy with concomitant increase in expression of muscle-specific
genes in isolated rat neonatal cardiomyocytes, GH did not have an
observable effect in this model. Similarly, Donath et al.
(17) reported that in cardiomyocytes from adult rats IGF-1, but not GH,
enhances myofibril development and down-regulates smooth muscle-
actin. In support of a direct (i.e. not secondary to
stimulation of circulating IGF-1) effect of GH on cardiac function is
the demonstration that ex vivo perfusion of the isolated rat heart with GH results in enhanced protein synthesis (21).
At present, it is not known if GH, acting through its cognate receptor
(growth hormone receptor (GHR)), has direct effects on the heart or if
some or all of the observed cardiovascular effects of GH administration
are secondary to an increase in circulating levels of IGF-1
and/or hemodynamic changes in vascular tone and blood pressure (22).
Recently, Lupu et al. (23) reported that in a knockout model
of GHR deficiency characterized by severe growth retardation, the
weight of the heart in the adult animal, although decreased in absolute
terms, was similar to controls when expressed relative to total body
weight. Moreover, relative levels of IGF-1 transcripts in the heart
also were unaffected. Thus, in this model of GHR deficiency, myocardial
growth and IGF-1 expression are implicitly independent of the GH/GHR
axis. However, the studies of Lupu et al. (23) do not
preclude a role for GH/GHR in normal growth and development of the
neonatal heart, including adaptive myocardial metabolism, characterized
by a transition from virtually exclusive utilization of glucose and
lactate in the fetus to fatty acids in the mature animal (24).
A major impediment to investigating the role of GH/GHR in the heart is
the lack of a suitable in vitro tissue culture model. We
have developed such a model by exploiting the strategy of recombinant adenovirus-mediated overexpression of GHR in neonatal rat
cardiomyocytes. Using this model, we demonstrate that GH stimulates
hypertrophy of the isolated cardiomyocyte, and these actions of GH seem
to be largely independent of changes in IGF-1 gene expression, findings that support an important independent role for the GH/GHR axis in
normal cardiac growth. In addition, GH alters the metabolic profile of
rat neonatal cardiomyocytes by inhibiting glucose uptake, stimulating
fatty acid uptake, and increasing protein synthesis, changes that
facilitate both postnatal cardiac growth and maturation of myocardial metabolism.
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EXPERIMENTAL PROCEDURES |
Isolation of Cardiomyocytes--
Neonatal ventricular myocytes
were prepared from hearts of 1-3-day-old Harlan Sprague-Dawley rats
using a previously described protocol (25, 26). Briefly, hearts were
removed and transferred to a 100-mm dish containing heparin solution
(1× Hanks' balanced saline solution, 0.8 µM
MgSO4, 20 mM HEPES, and 10 units of heparin). After dissecting hearts into two pieces with a razor blade, the tissue
was washed once with the heparin solution and incubated with gentle
rocking in a solution containing 1× Hanks' balanced saline
solution, 0.8 µM MgSO4, 20 mM HEPES, 2 µg/ml DNase, and 2 mg/ml trypsin. Tissue was
pipetted three times every 20-min with a 10-ml-wide mouth pipette, and
the supernatant was discarded at each time. Cardiomyocytes were
recovered afterward by alternating rocking and pipetting of tissue
every 5 min and pooled into a 50-ml tube containing 7 ml of fetal
bovine serum. Cells were centrifuged at 1000 rpm for 5 min and washed
once with cold DMEM containing 5% fetal bovine serum. Fibroblasts were
removed by plating (100-mm dishes) cells at 37 °C for 30 min in DMEM
containing 5% fetal bovine serum. Cardiomyocytes were recovered by
collecting the nonadherent cells and cultured in DMEM/F-12 medium
containing 5% charcoal-stripped calf serum (Sigma), 10 mM
HEPES, insulin-transferrin sodium selenite (5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selinium selenite, respectively), 10 mM glutamine, 30 µg/ml bromodeoxyuridine, and 10 µg/ml
gentamicin. Cells were plated on Pronectin
(BIOSOURCE)-coated tissue culture plates at a
density of 1 × 105 cells/cm2 and grown at
37 °C in 5% CO2. As assessed by staining with
antisarcomeric antibody (MF20; Developmental Studies Hybridoma Bank,
University of Iowa), these cell preparations typically contain greater
than 95% cardiomyocytes, form a nearly confluent monolayer by the
second day after plating, and beat spontaneously and synchronously.
After 24 or 48 h of incubation, cells were washed with serum-free
DMEM/F-12 culture medium and infected with recombinant adenovirus
expressing either the murine GHR (GHRAdlox) or the
-galactosidase
(
-galAdlox) gene.
Engineering and Production of Recombinant Adenoviral
Vectors--
The second generation shuttle vector pADLOX was modified
to contain either the
-galactosidase (
-galAdlox) or the murine GHR cDNA (GHRAdlox). The in vitro coupled
transcription-translation technique (Promega) was used to confirm that
the plasmids coded for proteins of appropriate molecular weight (data
not shown). The production of the replication-deficient adenoviral
vectors expressing either the
-galactosidase or GHR cDNA was
achieved by a protocol based on the Cre-lox principle for efficient and relatively quick identification of recombinant viral particles (27).
The adenovirus was plaque-purified, and a cesium chloride preparation
was used for the experiments.
Scatchard Analysis--
The number of cell surface receptors was
determined by competition binding using 125I-labeled human
GH. 1 × 106 aliquots of neonatal cardiomyocytes
(uninfected and infected at MOI of 12.5 plaque-forming units/cell for
48 h) were washed with binding buffer (25 mM HEPES (pH
7.4), 125 mM NaCl, 4 mM KCl, 1 mM
CaCl2, 1.5 mM MgCl2, 2 mM KH2PO4, and 1% human serum
albumin), incubated with 50,000 cpm 125I-human GH
(PerkinElmer Life Sciences) and various concentrations of unlabeled GH
for 90 min in binding buffer before centrifugation through 100 µl of
dibutyl phthalate. Specific binding was determined by subtracting the
amount of 125I-human GH bound in the presence of excess
unlabeled rGH (National Hormone and Pituitary Program, National
Institutes of Health), and the binding characteristics were analyzed
using a computer program (GraphPad).
Immunofluorescence and Planimetry--
After 24 h of
adenoviral infection, cells were rinsed with phosphate-buffered saline,
fixed in 4% paraformaldehyde, and permeabilized with 0.1% Triton
X-100. Following three phosphate-buffered saline washes, the chamber
slides were incubated in 0.1% bovine serum albumin for 30 min to block
nonspecific sites. Subsequently, the cells were covered with either
anti-GHR antibody (GHR-2; specific for the cytoplasmic region of the
GHR molecule and does not cross-react with GH-binding protein
(28)) or antisarcomeric myosin antibody (MF-20). Following incubation
for 1 h in the dark with the primary antibody, the cells were
washed four times with phosphate-buffered saline prior to exposure to
Texas Red-conjugated secondary antibodies. A Zeiss Axiovert 135 microscope equipped with a video camera system was used to capture
images and to determine cell size, estimated by measuring the area to
which individual MF-20-positive cells attached. 20-45 randomly
selected cells from each group (
-galAdlox- or GHRAdlox-infected,
with and without exposure to rGH) were chosen for such analysis, which
was conducted by an operator blinded to the identity of the cells being
examined. The data gathered were analyzed using ImagePro software.
Isolation of RNA--
GHRAdlox- or
-galAdlox-infected cells
were incubated with or without rGH for certain time periods. Cells were
washed once with ice-cold phosphate-buffered saline, and total RNA was
prepared by the Tri-Reagent method and quantitated by absorbance
at 260 nm. The integrity of the RNA and the accuracy of the
spectrophotometric determinations were confirmed by visual inspection
of the ethidium bromide-stained 28 and 18 S ribosomal RNA bands after
agarose-formaldehyde gel electrophoresis.
Ribonuclease Protection Assay for IGF-1--
IGF-I gene
expression was studied by RNase protection assay as previously reported
(29, 30). Briefly, 10-50-µg aliquots of total RNA were hybridized to
32P-labeled riboprobes generated from templates containing
exon 4 of the rat IGF-I gene (a 376-base pair
Sau3AI-EcoRI fragment) or 18 S rRNA (Ambion).
Protected probes were denatured, electrophoresed on an 8%
polyacrylamide gel, and exposed to X-Omat AR film for 1-2 days. The
protected bands corresponding to IGF-I mRNA and 18 S rRNA were
scanned using an Agfa Arcus II scanner, and densitometric analysis was
performed using the MacBAS version 2.31 computer program (Fuji Photo
Film Co., Tokyo, Japan). The level of IGF-I mRNA was normalized for
the amount of 18 S rRNA and analyzed as relative abundance to untreated
control samples.
Real Time Semiquantitative RT-PCR Assay--
Real-time
quantitative RT-PCR using the ABI Prism 7700 Sequence Detection System
(PE Biosystems, Foster City, CA) was carried out using established
protocols (31-35). The primers (synthesized by Life Technologies,
Inc.) and TaqMan probes (synthesized by Applied Biosystems) for the
quantitation of the GHR, IGF-1, fatty acid transport protein (FATP),
c-fos, and ANF transcripts were designed using the primer
design software Primer Express (Applied Biosystems) (Table
I). The primers and TaqMan probe for 18 S rRNA were purchased from a commercial vendor (PerkinElmer Life Sciences). The 18 S probe was labeled with reporter fluorescent dyes
VIC and the GHR, IGF-1, FATP, c-fos, and ANF probes with 6-carboxyfluorescein. The relative efficiencies of the GHR and IGF-1 primers/probe sets and the 18 S primer/probe pair were tested by
subjecting serial dilutions of a single RNA sample from each of the
tissues analyzed to real time RT-PCR analysis. The plot of log input
versus
CT was <0.1, which satisfies the previously established criterion for equivalence of efficiency of
amplification (34). CT, or threshold cycle,
represents the PCR cycle at which an increase in reporter fluorescence
above a base-line signal can first be detected, and
CT refers to the difference between the threshold
cycles for the target and the reference. After confirming that the
efficiencies of amplification of the gene of interest (e.g.
GHR) and 18 S transcripts were approximately equal, the amount of the
transcripts for the specific gene relative to the 18 S transcript was
determined by using the comparative CT (separate
tube) method (34). The comparative CT method is
similar to the conventional standard curve method, except it uses
arithmetic formulas to achieve the same result for relative quantitation. The amount of target, normalized to an endogenous reference and relative to a calibrator is given by the following formula: -fold induction = 2
CT, where

CT = (CT GI (unknown
sample)
CT 18 S (unknown sample))
(CT GI (adult heart)
CT 18 S (adult heart)); GI represents the gene of interest
(e.g. GHR). Briefly, 2-ng aliquots of total RNA were
analyzed using the One-Tube RT-PCR protocol (Applied Biosystems).
Following reverse transcription at 48 °C for 30 min, the samples
were subjected to PCR analysis using the following cycling parameters:
95 °C for 10 min; 95 °C for 15 s
60 °C for 1 min for
40 cycles. Each sample was analyzed in triplicate in individual assays
performed on two or more occasions.
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Table I
Sequence of primer and probe sets for TaqMan RT-PCR assay
5'-End reporter dyes were FAM (6-carboxylfluorescein) and VIC
(proprietary; Applied Biosystems). The quencher fluorescent dye at the
3'-end was TAMRA (6-carboxytetramethylrhodamine) for all probes.
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Western Blot Analysis--
For biochemical activation
experiments, serum-starved cardiomyocytes were treated with rGH
(500-1250 ng/ml) or vehicle for the indicated duration. The pelleted
cells were solubilized for 15 min at 4 °C in fusion lysis buffer
(1% (v/v) Triton X-100, 150 mM NaCl, 10% (v/v) glycerol,
50 mM Tris-HCl (pH 8.0), 100 mM NaF, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 10 mM benzamidine, 10 µg/ml aprotinin), as indicated. After centrifugation at 15,000 × g for 15 min at 4 °C, 10-15-µg aliquots of the
detergent extracts were resolved under reduced conditions by
SDS-polyacrylamide gel electrophoresis. Western transfer of proteins
and blocking of Hybond-ECL membranes (Amersham Pharmacia Biotech) with
2% bovine serum albumin were performed as previously described
(36-39). Equality of loading and efficiency of transfer were assessed
by Ponceau staining of the nitrocellulose filters. Membranes were
immunoblotted with the indicated dilutions of antibodies against total
MAPK (1 µg/ml), phospho-MAPK (1:20,000), phosphotyrosine-STAT5
(1:5000), phospho-Akt (1:1000), anti-JAK2 (1:1000), anti-GHR (1:1000),
anti-Glut1 (1:2500), anti-Glut4 (1:2500), or anti-
-actin (1:500).
Detection by ECL detection reagents (Amersham Pharmacia Biotech) and
stripping and reprobing of blots were accomplished according to the
manufacturer's suggestions.
Antibodies--
Anti-GHR used for immunostaining was a gift of
Dr. F. Talamantes (28). Antisarcomeric myosin antibody (MF-20) was
obtained from the Developmental Studies Hybridoma Bank (University of
Iowa). Antibodies against Glut1 and Glut4 have been described
previously (39), and anti-actin antibody was purchased from Sigma.
Anti-phosphotyrosine monoclonal antibody (4G10) and anti-MAPK
affinity-purified rabbit antibody (directed at residues 333-367 of rat
ERK1; recognizes both ERK1 and ERK2) were purchased from Upstate
Biotechnology, Inc. (Lake Placid, NY). Anti-activated (anti-phospho-)
MAPK affinity-purified rabbit antibody (recognizing the dually
phosphorylated Thr183 and Tyr185 residues that
correspond to the active forms of ERK1 and ERK2) was purchased from
Promega (Madison, WI). Anti-phospho-Akt (specifically recognizing Akt
phosphorylated at Ser473) affinity-purified rabbit
antibodies were purchased from New England Biolabs (Beverly, MA).
Rabbit anti-phosphotyrosine-STAT5 polyclonal antibody (raised against a
phosphopeptide surrounding phosphorylated Tyr694 of murine
STAT5A, which is conserved in both STAT5A and STAT5B) was obtained from
Zymed Laboratories Inc. (San Francisco, CA). The
rabbit polyclonal serum, anti-JAK2AL33 (referred to herein as anti-JAK2), has been described previously (40).
Anti-GHRcyt-AL47 (referred to as anti-GHR in immunoblotting
experiments) is a new antibody developed in the Frank laboratory. This
rabbit serum was raised against a bacterially expressed N-terminally His-tagged fusion protein incorporating the entire cytoplasmic domain
of the human GHR (residues 271-620) (41). The cDNA encoding this
fusion was created by PCR in the pET vector system (Novagen) (PCR
primer sequences available upon request). Molecular biology techniques,
cDNA sequencing for verification of fidelity, bacterial fusion
protein expression, and preparation of
Ni2+-agarose-purified fusion protein were performed
according to our previous methods (36, 37, 40, 42) and the
manufacturer's suggestions.
Quantification of Glucose Transport--
Glucose uptake was
assayed at 37 °C as 2-deoxy-D-glucose
(2-G-3H-labeled) internalization. Briefly,
cardiomyocytes exposed to either rGH (1250 ng/ml) or the vehicle were
incubated in DMEM (1 g/liter glucose) containing 1 µci/ml
2-deoxy-D-glucose (2-3H-G-labeled) (PerkinElmer
Life Sciences). At specific time points, cultures were removed from the
incubator, and cytochalasin B (250 µM final
concentration) was added to halt further hexose uptake. The monolayer
was then washed three times with ice-cold PBS, the washed
cardiomyocytes were solubilized by exposure to 0.5 N NaOH for 10 min and neutralized with equal volume of 0.5 N HCl,
and aliquots were taken for measurement of 3H activity by
scintillation counting and estimation of protein content by the
Bradford assay. The glucose uptake was calculated by subtracting
nonspecific cell-associated radioactivity measured by including 250 µM cytochalasin B in a parallel series of cultures during
the incubation with 2-deoxy-D-glucose
(2-3H-G-labeled). The basal level of glucose transport in
cells transduced with recombinant adenovirus ranged between 2 and 8.1 nmol/mg of protein.
Incorporation of Leucine
(L-3,4,5-3H-Labeled)--
Infected or
uninfected cardiomyocytes were incubated in DMEM/F-12 medium containing
12.5 µCi/ml L-[3,4,5-3H]leucine
(PerkinElmer Life Sciences) with or without rGH (0.5-1250 ng/ml) for
24 h. At the end of incubation, cells were washed two times with
ice-cold PBS and incubated with 10% trichloroacetic acid at 4 °C
for 10 min. After washing with ice-cold ethanol, the cells were
solubilized in 0.5 N NaOH for 10 min and then neutralized with an equal amount of 0.5 N HCl. The uptake of
3H was determined by scintillation counting.
Palmitic Acid Uptake Assay--
Palmitate uptake studies were
performed in infected and uninfected cardiomyocytes that were treated
or untreated with rGH (0.5-1250 ng/ml) for 4 h. Albumin-bound
palmitate was prepared by dissolving unlabeled sodium palmitate
(hexadecanoic acid; Sigma) and [9,10-3H]palmitic acid
(American Radiolabeled Chemicals) in 10 ml of water, to give a
concentration of 320 µM (60 µCi/ml). Fatty acid-free bovine serum albumin was added to the above solution to obtain a
palmitate/PBS ratio of 4.0. This stock solution was diluted 1:8 with
PBS before adding to the cells, and the incubation was carried out for
2 min. Palmitate uptake was terminated by adding cold PBS containing
0.1% PBS and 200 µM phloretin. Cells were then washed
five times with cold PBS containing 0.1% bovine serum albumin,
solubilized with 0.5 N NaOH, and then neutralized with 0.5 N HCl. Palmitate uptake was determined by scintillation counting.
Data Analysis--
Data are presented as either mean ± S.D. or mean ± S.E. as indicated. Statistical differences between
groups were determined by ANOVA. p values equal to or less
than 0.05 were considered significant.
The study protocol was approved by the Children's Hospital of
Pittsburgh Animal Care and Use Committee, and the animals received humane care in compliance with the National Research Council's criteria as outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Institutes of Health.
 |
RESULTS |
Dysregulation of Expression of GHR during Isolation of Neonatal
Cardiomyocytes--
Isolated cardiomyocytes from the neonatal rat
heart are a standard and accepted cell culture model for studying
mammalian cardiac cell physiology that has been used to examine
the role of the GH/IGF-1 system in the heart (20). To ascertain if
cultured cardiac cells are an appropriate model in which to study the
effects of GH, qualitative RT-PCR was performed using RNA isolated
directly from rat heart or from cultured cardiomyocytes (Fig.
1A). Comparison of GHR
transcript levels in isolated neonatal cells (Fig. 1A, lane 9) versus that found in neonatal
whole heart (Fig. 1A, lane 7)
indicates that either during isolation and/or culture, GHR mRNA
levels decrease significantly in isolated cardiomyocytes. To confirm
these qualitative data, a quantitative analysis was next performed
using a fluorescent 5'-nuclease (TaqMan) assay. These data confirmed
that the expression of GHR mRNA was decreased in isolated rat
neonatal cardiomyocytes by ~70-80% compared with either the intact
neonatal or the adult heart, respectively (Table II). Indirect immunofluorescence
microscopy revealed that most of the endogenous GHR in the cultured
cardiomyocyte was perinuclear and not in the cellular membranes, as
seen in tissues, including the heart, in the intact animal (Fig.
1B) (43, 44). Thus, the dual findings of low levels of GHR
mRNA and the aberrant localization of the GHR protein in these
cells may explain the observation of others that GH treatment has
little effect on the rat primary neonatal cardiomyocyte in culture (17,
20). Furthermore, adult whole heart expresses about 35% more GHR
transcripts than the neonatal heart (Fig. 1A and Table II),
thus increasing the possibility that GH has direct effects on adult
cardiomyocytes in vivo.

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Fig. 1.
A, RT-PCR measurement of GHR mRNA
levels in adult and neonatal rat heart and neonatal rat isolated
cardiomyocytes. Expression of GHR transcripts in rodent heart or liver
is shown. RT-PCR was performed using total RNA from no RT control-adult
rat liver (lane 1); adult mouse liver
(lane 2); adult mouse heart (lane
3); adult rat liver (lane 4); adult
rat heart (lane 5); adult rat primary
cardiomyocytes (lane 6); neonatal rat heart
(lane 7); neonatal rat noncardiomyocytes
(lane 8); and neonatal rat primary cardiomyocytes
(lane 9). A 1-µg aliquot of total RNA was split
to amplify either rodent GHR (upper panel) or
GAPDH transcripts (bottom panel). Note the low
levels of GHR in isolated neonatal cardiomyocytes (lane
9) compared with adult heart tissue (lane
5) or neonatal rat heart (lane 7).
B, pattern of expression of GHR in cultured cardiomyocytes.
Isolated primary rat neonatal cardiomyocytes were cultured for 48 h before fixation in 4% paraformaldehyde. Cells were analyzed by
indirect immunofluorescence using primary antibody specific for the
cytoplasmic region of the GHR (GHR-2). Note perinuclear localization of
GHR fluorescence (arrow) with little membrane-associated
fluorescence.
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Table II
TaqMan RT-PCR assay for relative quantification of GHR transcripts in
adult heart, neonatal heart, and isolated neonatal cardiomyocytes
AH, adult rat heart RNA; NH, neonatal rat heart RNA; NCM, isolated
neonatal cardiomyocyte RNA. Results represent the average of at least
three independent measurements. , p < 0.05 compared with NH.
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Adenovirus-mediated Overexpression of GHR--
To determine
whether cultured cardiomyocytes could be made sensitive to the effects
of GH, we explored means to increase the expression of GHR in rat
neonatal cardiomyocytes. Pilot experiments revealed that strategies to
obtain a homogenous population of cardiomyocytes overexpressing GHR,
such as a selection process based on co-transfection of the plasmid
pHook-2 (Invitrogen), resulted in poor yields. Hence, we chose to
employ an alternate strategy using recombinant adenovirus (45, 46). The
second generation shuttle vector pADLOX was modified to contain either the
-galactosidase (
-galAdlox) or the murine GHR cDNA
(GHRAdlox) under the control of the cytomegalovirus promoter. The
production of replication-deficient adenoviral vectors expressing
either
-galactosidase or GHR cDNA was achieved by a protocol
based on the Cre-lox principle (27). A multiplicity of infection of
~12.5 plaque-forming units/cell was routinely used in this study
because pilot experiments determined this multiplicity of infection to be the upper limit of tolerance of the isolated cardiomyocytes to
adenoviral infection. Analysis of cell lysates by Western blot with an
antiserum against the GHR cytoplasmic domain shows the expected GHR
protein product (lower panel of Fig.
2A and upper panel of Fig. 2B). The number of cell surface
receptors was determined by competition binding using
125I-labeled human GH. Whereas there was no specific
binding with noninfected cardiomyocytes, 140,000 ± 23,000 receptors/cell with a Kd of 4.9 ± 1.1 nM were detected on the infected cardiomyocytes.

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Fig. 2.
GH stimulates GH-dependent
post-receptor signaling pathways in GHR-overexpressing rat neonatal
cardiomyocytes. A and B, isolated rat neonatal
cardiomyocytes overexpressing either -galactosidase or GHR were
stimulated with rGH (500 ng/ml) or vehicle (0 min) for the indicated
durations. Detergent-solubilized proteins were resolved by
SDS-polyacrylamide gel electrophoresis and sequentially immunoblotted
with the indicated antibodies. In A, the position of the
prestained 97-kDa molecular mass marker is indicated. P-GHR,
P-JAK2, and P-STAT5, tyrosine-phosphorylated GHR,
JAK2, and STAT5, respectively. P-Akt, serine-phosphorylated
(activated) Akt; P-MAPK, dually (threonine and tyrosine)
phosphorylated (activated) ERK-1 and -2, as described under
"Experimental Procedures." The abundance of total MAPK is indicated
in the bottom panel. The positions of JAK2 and
GHR present in the extracts are indicated also. The experiments shown
are representative of two such experiments.
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GH-GHR Signaling Pathways in Isolated Cardiomyocytes--
GH
signaling is initiated by ligand-induced GHR dimerization and
activation of the receptor-associated tyrosine kinase, JAK2 (38, 47,
48). This results in tyrosine phosphorylation of JAK2, the GHR, and
other intracellular proteins (see Refs. 49 and 50 and references
therein). With the exception of calcium influx (51), the
GH-dependent activation of intracellular signal cascades,
such as the STAT5, phosphatidylinositol 3-kinase/Akt, and MAPK pathways
requires JAK2 activation (49, 50). To determine if GH signaling in our
in vitro model system was similarly associated with
intracellular tyrosine phosphorylation, cells stimulated with GH or
vehicle were detergent-solubilized, and proteins resolved by
SDS-polyacrylamide gel electrophoresis were immunoblotted with anti-phosphotyrosine antibodies (Fig. 2A, upper
panel). Stimulation of isolated cardiomyocytes transduced
with the GHR-overexpressing virus with GH for 15 or 30 min resulted in
the appearance of two prominent tyrosine-phosphorylated bands in the
100-125-kDa range, which were not detected in extracts of the cells
transduced with the
-galactosidase overexpressing virus
(lanes 4-6 versus lanes 1-3). By analogy to our previous studies of rodent cells
(36, 38), these GH-induced tyrosine phosphoproteins (a sharp band designated by an arrow and a diffuse, more rapidly migrating
band designated by a bracket) probably include JAK2 and the
GHR. To further their identification, the nitrocellulose filter was
sequentially stripped and reprobed with anti-JAK2AL33 (Fig.
2A, middle panel) and
anti-GHRAL47 (Fig. 2A, lower
panel) sera. As expected, endogenous JAK2 (arrow)
was detectable at similar levels in each sample, while GHR
(bracket that includes the fully glycosylated and faster migrating incompletely glycosylated GHR forms (52)) was present only in
the GHR-overexpressing cells. The tyrosine phosphoproteins present in
lanes 5 and 6 of the upper
panel exactly comigrated with JAK2 and the fully
glycosylated GHR and are thus designated P-JAK2 and P-GHR, as
indicated. These data strongly suggest that GH acutely promotes JAK2
and GHR tyrosine phosphorylation in the GHR-reconstituted cardiomyocytes.
To further characterize signaling pathways downstream of JAK2 activated
by GH in our cardiomyocyte model system, we performed a similar
experiment in which
-galactosidase- or GHR-overexpressing cardiomyocytes were treated with vehicle or GH for 15 min. Extracted proteins were separated by SDS-polyacrylamide gel electrophoresis and
sequentially immunoblotted with activation state-specific antibodies
that detect the phosphorylated forms of STAT5, Akt, and MAPK (ERK1 and
ERK2) (Fig. 2B). We observed robust GH-induced phosphorylation of each signaling molecule in the GHR-overexpressing cells, while the
-galactosidase-overexpressing cells exhibited a
more modest degree of activation of STAT5 and Akt phosphorylation and
minimal MAPK phosphorylation (with a higher basal degree of MAPK
activation in these cells). Similarity of loading was demonstrated by
assaying for total MAPK (Fig. 2B, bottom
panel). In other experiments (not shown),
GH-dependent activation of the STAT5, Akt, and MAPK pathways was consistently observed in GHR-transduced cells, in contrast
to inconsistent and weak responses in the
-galactosidase-overexpressing cells. These data suggest that the
canonical pathways of GH-GHR signaling cascades are reliably intact in
cardiomyocytes overexpressing the GHR.
GH Modulates Metabolic Parameters in Isolated
Cardiomyocytes--
GH is known to alter metabolic parameters of
GH-responsive tissues. Having established that the recombinant
adenovirus-infected cardiomyocytes expressed GHR, we next tested the
effect of GH on metabolic parameters such as incorporation of amino
acids, fatty acids, and glucose uptake of cardiomyocytes in this model. GH did not alter the uptake of any of the metabolites in nontransduced or
-galactosidase-overexpressing adenovirus-transduced
cardiomyocytes. However, in GHR-overexpressing neonatal cardiomyocytes,
GH increased the incorporation of
L-[3,4,5-3H]leucine in a
dose-dependent manner: 127 ± 7, 144 ± 14, and
163 ± 28% at doses of 0.5, 50, and 1250 ng/ml, respectively
(mean ± S.E.; n = 3) (Fig.
3). Likewise, GH increased the uptake of [9,10-3H]palmitic acid, although a statistically
significant effect (151 ± 10%) was observable only at a GH
concentration of 1250 ng/ml (Fig. 4). In
contrast, the uptake of 2-deoxy-D-glucose
(2-G-3H-labeled) was inhibited by 60 ± 11%
after 4 h of exposure to a GH concentration of 1250 ng/ml (Fig.
5).

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Fig. 3.
Stimulation of leucine uptake by GH in
GHR-overexpressing rat neonatal cardiomyocytes. GHR overexpressing
cardiac myocytes were incubated with 12.5 µci/ml leucine
(L-3,4,5-3H-labeled) and exposed to
varying concentrations of rGH (0, 0.5, 50, and 1250 ng/ml) for 24 h. Cellular uptake of leucine
(L-3,4,5-3H-labeled) was measured by
scintillation counting at the end of the incubation period. Results
represent mean ± S.E. of three separate experiments. ,
p < 0.05 compared with GH (0 ng/ml) control by
ANOVA.
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Fig. 4.
Stimulation of fatty acid uptake by GH in
GHR-overexpressing rat neonatal cardiomyocytes. GHR-overexpressing
cardiomyocytes were incubated with 0, 50, 500, or 1250 ng/ml of rGH for
4 h. Fatty acid uptake was measured 2 min after the addition of a
solution containing 40 µM palmitic acid, 10 µM bovine serum albumin, and tracer
[9,10-3H]palmitic acid. Fatty acid uptake was estimated
by scintillation counting at the end of the incubation period. Results
represent mean ± S.E. of three separate experiments. ,
p < 0.05 (ANOVA) compared with in the absence of
GH.
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Fig. 5.
Inhibition of glucose uptake by GH in
GHR-overexpressing rat neonatal cardiomyocytes. GHR-overexpressing
cardiomyocytes were incubated in DMEM containing 1 g/liter glucose plus
1 µCi/ml 2-deoxy-D-glucose
(2-G-3H-labeled) in the absence (open
bar) or presence (solid bar) of rGH
(1250 ng/ml) for 10 min, 1 h, or 4 h.
2-deoxy-D-glucose (2-G-3H-labeled) uptake was
measured by scintillation counting at the end of the respective
incubation period. Results represent mean ± S.E. of three
separate experiments. , p < 0.05 (ANOVA) compared
with in the absence of GH.
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To ascertain the possible mechanism for the impaired glucose uptake, we
next measured the levels of glucose transport proteins in these
samples. Western blot analysis of cell lysates determined that GH
decreased the levels of Glut1 by ~25-30% in GHR-overexpressing cardiomyocytes compared with cardiomyocytes transduced with the
-galactosidase adenovirus (Fig. 6,
A and B). In contrast, there was no change in the
levels of Glut4 transport protein in these samples. In addition,
estimation of hexokinase activity did not reveal any significant
changes in cells after exposure to GH (data not shown). These results
indicate that the GH-induced decrease in glucose uptake by
cardiomyocytes is in part due to alteration in levels of the Glut1
protein.

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Fig. 6.
Glut1 and Glut4 protein levels in GH-treated
GHR-overexpressing rat neonatal cardiomyocytes. A,
quantitative analysis of Glut1 and -4 protein expression.
-Galactosidase-overexpressing (open bars) or
GHR-overexpressing (solid bars) cardiomyocytes
were stimulated with 1250 ng/ml rGH for 4 h. Cell lysates were
collected from these two treatment groups. 20-µg aliquots of protein
lysate were analyzed for Glut1 or Glut4 expression by Western blot
analysis using anti-rat Glut1 or Glut4 antibodies. The blots were
reprobed for -actin to normalize for protein loading. The intensity
of signal was determined by densitometry quantification. Results
represent mean ± S.E. of three separate experiments. ,
p < 0.05 (ANOVA) compared with
-galactosidase-overexpressing cardiomyocytes. B, Western
blot analysis of Glut1 and Glut4 expression. Representative Western
blots demonstrating the 50-kDa Glut1, 48-kDa Glut4, and the actin bands
are shown.
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Fatty acid transport across membranes can be due to both diffusion and
carrier-mediated processes (53). Many proteins are thought to
participate in the transmembrane translocation of fatty acids. These
include fatty acid-binding proteins, fatty acid translocase, and fatty
acid transport proteins (54). Having demonstrated that GH increased the
uptake of fatty acids in the GHR-overexpressing cardiomyocytes, we used
the fluorescent 5'-nuclease (TaqMan) assay to measure the levels of
mRNA for one of the known FATPs in this model system (55). These
studies revealed that the GH increased the levels of mRNA for FATP
by ~50% in GHR-overexpressing cardiomyocytes in comparison with
-galactosidase-overexpressing cardiomyocytes (Fig.
7).

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Fig. 7.
GH stimulates FATP gene expression in
GHR-overexpressing rat neonatal cardiomyocytes. -Galactosidase
(open bar) or GHR (solid
bar) overexpressing cardiomyocytes were exposed to rGH (500 ng/ml) for 4 h, and total RNA was extracted for analysis of FATP
gene expression using a fluorescent 5'-nuclease (TaqMan) assay and
employing the comparative CT method. Expression of
the FATP gene was normalized for expression of the 18 S gene. Results
represent mean ± S.E. of three separate experiments. ,
p < 0.05 (ANOVA) compared with
-galactosidase-overexpressing cardiomyocytes.
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GH Stimulates Hypertrophy of Isolated Cardiomyocytes--
IGF-1 is
known to induce hypertrophy of the isolated cardiomyocyte, and previous
reports have concluded that GH does not have a similar effect on these
cells (20). We reexamined the issue of direct effects of GH in our
model of GHR-overexpressing cardiomyocytes. For this purpose, we
measured the area of cells exposed to GH or the vehicle only. These
experiments revealed that cardiomyocytes transduced with the
-galactosidase-overexpressing adenovirus did not undergo changes in
cell size on exposure to rGH (GH(
) versus GH(+), 1083 ± 36 versus 1087 ± 58, mean ± S.E.;
p > 0.05 by ANOVA). In contrast, exposure to rGH (500 ng/ml) for 24 h resulted in a significant increase in the size of
the cardiomyocyte transduced with the GHR-overexpressing adenovirus
(GH(
) versus GH(+), 947 ± 48 versus
1651 ± 64 µm; p < 0.01 by ANOVA).
Cardiac hypertrophy is associated with characteristic
changes in gene expression. Thus, it is well established that the
expression of early response genes (e.g. c-fos,
c-myc, c-jun, egr-1) are activated within 30-60 min after exposure to a hypertrophic stimulus. Additionally, "late" markers of cardiac hypertrophy
(e.g. ANF, myosin light chain 2,
-skeletal actin,
-myosin heavy chain) are also variably activated in various forms of
cardiac hypertrophy. We utilized the TaqMan assay to profile the
GH-dependent expression of representative genes from the
"early" (i.e. c-fos) and from the "late"
(i.e. ANF, myosin light chain 2,
-skeletal actin) response group of genes in our model system (Fig.
8). Our results indicate that in
GHR-overexpressing cells, GH elicited a robust (500-600%) increase in
expression of c-fos mRNA and a modest (40-60%) increase in expression of myosin light chain 2 and
-skeletal actin
mRNA. In contrast, GH did not alter the expression of ANF mRNA
in these cells.

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Fig. 8.
GH-induced alterations in genetic markers of
hypertrophy in GHR-overexpressing rat neonatal cardiomyocytes.
GHR-overexpressing cardiomyocytes were exposed to rGH (500 ng/ml) for
the indicated time periods, and total RNA was extracted for analysis of
c-fos, myosin light chain 2, skeletal -actin, or ANF gene
expression using fluorescent 5'-nuclease (TaqMan) assays and employing
the comparative CT method. Expression of the
specific mRNA was normalized for expression of the 18 S gene.
Results represent the mean ± S.E. of three independent
experiments. , p < 0.05, not significant
(ns) = p > 0.05 (ANOVA) compared with
cells exposed to vehicle only.
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GH's Actions on Isolated Cardiomyocytes Are Independent of
IGF-1--
At the cellular level, the actions of GH can be either
attributed to direct effects of GH or due to indirect effects, since GH
induces the synthesis of IGF-1 in many tissues and IGF-1 has potent
actions in these tissues (56). To determine if the effects of GH
observed in our model system were due to direct effects of GH or due to
stimulation of IGF-1 synthesis, we estimated the levels of IGF-1
mRNA by ribonuclease protection and fluorescent 5'-nuclease
(TaqMan) assays. The RPA analysis revealed that there was no change in
IGF-1 levels in response to GH stimulation for 24 h (Fig.
9A). To exclude the
possibility that changes in IGF-1 could have preceded the observed
effects of GH on the metabolic parameters and cell size, we used the
fluorescent 5'-nuclease (TaqMan) assay to investigate the temporal
profile of IGF-1 in our model system. These data reveal that, in
concert with the RPA data, there are no significant changes in IGF-1
mRNA levels in these cells in response to GH stimulation for 15 min, 60 min, or 4 h (Fig. 9B).

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Fig. 9.
GH-induced IGF-1 gene expression in
GHR-overexpressing rat neonatal cardiomyocytes. A,
ribonuclease protection assay. RNA was isolated from neonatal
cardiomyocytes (uninfected (lanes 2 and
3), infected with -galactosidase-overexpressing
adenovirus (lanes 4 and 5), or
infected with GHR-overexpressing adenovirus (lanes
6 and 7)), and the cells were either exposed for
24 h to rGH (500 ng/ml) (lanes 3,
5, and 7) or vehicle only (lanes
2, 4, and 6). Lane
1, size markers; lanes 8 and
9, control RNA from adult rat heart (lane
8) or liver (lane 9). 18 S RNA was
also assayed to ensure equality of RNA loading. Three protected bands
are visible: a major band of 224 bases and a minor band of 100 bases of
IGF-Ia mRNA (30), both being specific protections to different
regions of the probe, and an 80-base band corresponding to the 18 S
rRNA. Densitometric analysis of the protected bands confirmed the
absence of effect of GH on IGF-1 mRNA expression in GHR
overexpressing cells (lane 6 compared with
lane 7). A, fluorescent 5'-nuclease
assay. -Galactosidase-overexpressing (open
bars) or GHR-overexpressing (solid
bars) cardiomyocytes were exposed to rGH (500 ng/ml) for the
indicated time periods, and total RNA was extracted for analysis of
IGF-1 gene expression using a fluorescent 5'-nuclease (TaqMan) assay
and employing the comparative CT method. Expression
of the IGF-1 gene was normalized for expression of the 18 S gene.
Results represent mean ± S.E. of three separate experiments. Not
significant (ns) = p > 0.05 (ANOVA)
compared with -galactosidase-overexpressing cardiomyocytes.
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DISCUSSION |
We demonstrate that GH has effects on the isolated neonatal
cardiomyocyte expressing GHR. Our results indicate that GH causes hypertrophy of the cardiomyocyte and alters its metabolic profile by
decreasing glucose uptake while increasing fatty acid uptake and
protein accretion rate. Both the morphological and metabolic effects
are not temporally associated with changes in IGF-1 gene expression in
these cells.
It is claimed that IGF-1 has direct actions on cardiomyocytes, whereas
GH does not (17, 20). In this report, we demonstrate that the process
of isolation and culture of neonatal cardiomyocytes results in loss of
expression of GHR mRNA and aberrant localization of the GHR
protein. In contrast to this loss of expression of GHR mRNA, IGF-1
receptor mRNA is increased during the process of isolation and
culture (17). Thus, these experimental artifacts could be responsible
for the finding that these cells respond to IGF-1 but are unresponsive
to GH (20). Our data indicate that the strategy of using recombinant
adenovirus-mediated overexpression of GHR can be used to establish an
in vitro model of isolated cardiomyocytes that respond to
GH. It is noteworthy that in our model system we did observe some
GH-dependent effects in cardiomyocytes transfected with the
-galactosidase-overexpressing adenovirus. The only consistent
response following GH stimulation of cardiomyocytes not overexpressing
GHR was an increase in phospho-STAT5. This effect possibly reflects
signaling through GHR or prolactin receptors that remained intact
during the isolation and culture. Activation of phospho-Akt and
c-fos was also detectable in the
-galactosidase-overexpressing cells. However, these effects were
minimal and inconsistent, possibly reflecting variations in harvesting
and culture of these cells.
Whereas the actions of GH to alter the metabolic parameters in various
cell types are well established, the current report is the first to
demonstrate effects of GH on the metabolic parameters of the isolated
cardiomyocyte. In general, the effects of GH on carbohydrate metabolism
can be grouped into acute and chronic effects. The acute effects of GH,
commonly referred to as insulin-like actions, include decrease in blood
glucose concentration as well as stimulation of glucose uptake by
muscle and isolated adipocytes in vitro. In contrast, the
chronic effects of exposure to GH include increase in blood glucose
concentration, inhibition of glucose uptake, and insulin resistance
(16). Our results parallel observations in fat (57) and skeletal muscle
(58) that demonstrate that GH decreases basal glucose transport via a
decline in GH-induced Glut1 mRNA without affecting total cellular
content of Glut4 protein. However, since we only analyzed total
cellular Glut4, the possibility that alterations in subcellular
localization of Glut4 could contribute toward GH-induced perturbations
in insulin-responsive glucose transport cannot be excluded (59). Our
results indicate that GHR increases in the adult as compared with the
neonatal heart and that GH can increase the fatty acid uptake by
cardiomyocytes expressing GHR. In higher mammals, the ability of
cardiomyocytes to transport fatty acids increases with age, and there
is an adaptive transition from glucose to fatty acids as the preferred
oxidative fuel for the mammalian heart during maturation from the fetus to the adult (24). Likewise, in the rodent there is a 3-fold increase
in fatty acid oxidative rates of the heart muscle during maturation
from the neonate to the adult and a contemporaneous increase in
expression of fatty acid transport proteins (54, 60). Since GHR
expression increases postnatally, we postulate that this ontogenic
profile of expression of GHR in the heart plays a role in the
transition of metabolism of the mammalian heart, from dependence on
glucose in the fetus to preferential use of fatty acids postnatally.
Cardiac hypertrophy is a hallmark of GH excess in both animal models
and humans (1). In contrast to prior studies that concluded that GH
does not stimulate hypertrophy of cardiomyocytes, our results indicate
that GH does induce hypertrophy of isolated cardiomyocytes. Switches in
isoform expression of actin and myosin are observed with many forms of
cardiac hypertrophy, with each hypertrophic stimulus eliciting its
characteristic profile of actin/myosin isoforms. In general, cardiac
hypertrophy is associated with induction of fetal isoforms
(i.e. skeletal
-actin,
-myosin heavy chain),
expression of other fetal proteins (i.e. ANF), and up-regulation of constitutively expressed genes (i.e. myosin
light chain 2, cardiac
-actin) (61). In the current model system, GH
increased the expression of c-fos, myosin light chain 2, and skeletal
-actin but did not alter the expression of ANF. It is of
interest to note that combined administration of IGF-1 and GH in the
intact animal failed to alter expression of skeletal
-actin or ANF
gene expression (62). Similarly, IGF-1 decreased ANF gene expression in
isolated adult rat cardiomyocytes in culture (63). The differential
effects of GH on fetal gene expression with induction of skeletal
-actin but not of ANF support the hypothesis that activation of the
GH/IGF-1 axis induces a more physiological or nonpathological form of
cardiac hypertrophy (62). However, further studies are needed to
substantiate this hypothesis, and the current experimental paradigm of
adenovirus-mediated GHR-overexpression will facilitate further analysis
of the effects of GH/IGF-1 in both neonatal and adult cardiomyocytes.
The somatomedin hypothesis states that the anabolic and
growth-promoting effects of GH are principally mediated by stimulation of production of IGF-1 by the liver (endocrine action) and peripheral tissues (autocrine/paracrine action) (16). Contemporary studies using
techniques such as targeted disruption and conditional tissue-specific knockout of specific proteins involved in this pathway have begun to
reexamine this hypothesis (23, 64). Most recently, Lupu et
al. (23), using a total knockout model of GHR, demonstrate that
there must be independent and additive effects of GH and IGF-1 on
overall and specific tissue growth. Our results complement these
findings, since observed changes in the size of cardiomyocytes and
their metabolic profile in response to GH were not accompanied by
changes in IGF-1 gene expression. Furthermore, using radioimmunoassay, we were unable to detect changes in concentration of IGF-1 in the
conditioned medium (data not shown), suggesting that in this model system the effects of GH are, at least in part, independent of
IGF-1. It is also noteworthy that cardiac hypertrophy has been observed
in IGF-1 knockout mice (65). In these IGF-1 null mice, circulating
levels of GH are elevated, and one of the mechanisms resulting in
cardiac hypertrophy could be direct action of the elevated circulating
levels of GH on the heart, consistent with our results in the isolated
neonatal cardiomyocyte expressing GHR.
Lupu et al. (23) demonstrated that divergence of the growth
patterns of normal and GHR knockout animals occurs only after 2 weeks
of life. Our studies extend the observations of Lupu et al.
(23) by demonstrating that the relative lack of GH's effect on the
neonatal heart is, at least in part, due to the paucity of GHR in the
neonatal heart and that expression of GHR increases significantly in
the adult heart. Our model of neonatal cardiomyocytes transduced with
the GHR-expressing adenovirus thus tends to recapitulate the scenario
in the adult cardiomyocyte with respect to GHR expression. Hence, our
studies support the hypothesis that when the heart exhibits sufficient
GHR expression, as in the adult, it is a target of direct GH action.
This report describes a model based on rat neonatal cardiomyocytes.
Cardiomyocytes from immature animals (embryonic, fetal, and neonatal)
are easier to isolate and culture and thus have been widely used in
in vitro studies. Adult ventricular cardiomyocytes, however,
differ from immature cells. Thus, adult cells are morphologically
different in that they have a larger, more developed transverse tubular
system, grow more slowly in culture, and have a restricted potential
for differentiation (66). These differences should be taken into
account when interpreting the results of the present study obtained
with neonatal cardiomyocytes, and future studies carried out with adult
cardiomyocytes will shed light on the effects of GH on the adult heart.
It is noteworthy that our strategy of using adenovirus-mediated
overexpression of GHR will be suitable for use with adult
cardiomyocytes (67).
In summary, we have established a model suitable for examining the
effects of GH on cardiomyocytes expressing GHR. Our results indicate
that GH induces hypertrophy of these cells and causes alterations in
their metabolic profile independently of IGF-1. These studies add to
evidence that the effects of GH and IGF-1 on somatic growth may be
additive yet independently mediated and provide the first direct proof
of independent actions of GH on the heart.