Download Original Contributions Left Ventricular Insulin-like

25
Original Contributions
Left Ventricular Insulin-like Growth Factor I
Increases in Early Renal Hypertension
H&kan Wahlander, Jorgen Isgaard, Eva Jennische, and Peter Friberg
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
Increasing interest has been directed toward the possible role of trophically acting molecules as
modulators or initiators, or both, of myocardial hypertrophy. The aim of the present study was
to investigate the possible role of one such molecule, namely, insulin-like growth factor I, in
myocardial hypertrophy developed in response to renal artery stenosis. Two-kidney, one clip
Goldblatt hypertension was induced in Wistar rats weighing 180 g, and sham-operated animals
were used as controls. Blood pressure was increased as early as 2 days after clipping (133 ± 4
versus 116±4 mm Hg, p<0.05), and the increase persisted 4 and 7 days after clipping (148±6
versus 129±3 mm Hg, p<0.01 and 171±5 versus 139±3 mm Hg,p<0.01, respectively). Left
ventricular weight followed a similar pattern (373±7 versus 350±8 mg, NS, 415±11 versus
386±9 ing,/><0.01, and 466±11 versus 391 ±10 mg,/><0.01 at 2, 4, and 7 days after clipping,
respectively), but no changes in body weight between the groups were observed. Insulin-like
growth factor I messenger RNA (mRNA) was quantified using a solution hybridization assay.
After 4 days of renal hypertension, there was a significant increase in left ventricular
insulin-like growth factor I mRNA (2.0 • 10 1 8 ±0.48 • 10~18 versus 0.4 • 10"18±0.07 • 10"18
mol - fig DNA~'), which was no longer detectable 7 days after clipping. When insulin-like
growth factor I protein was visualized with immunohistochemistry, an enhanced abundance of
the protein was demonstrated in the left ventricle 4 as well as 7 days after clipping, and the
protein seemed mainly confined to the subendocardial layers where wall stress is highest In the
right ventricle, no changes in weight, insulin-like growth factor I mRNA, or protein could be
detected. In conclusion, two-kidney, one clip Goldblatt hypertension induced an increase in
insulin-like growth factor I mRNA as well as protein in the left but not in the right ventricle.
The principal localization of the increase in insulin-like growth factor I to the subendocardial
layers of the left ventricle suggests wall stress as the most probable initiating stimulus for the
enhanced synthesis of insulin-like growth factor L (Hypertension 1992;1925-32)
W
hen challenged by a chronically increased
systemic pressure load, the heart responds with an adaptive left ventricular
hypertrophy to normalize wall stress and myocardial
oxygen consumption (see Friberg1). However, the
precise mechanism for transforming the mechanical
stimuli of increased pressure or volume work into the
hypertrophic response is not known. In addition to
mechanical stimuli, different trophic factors such as
catecholamines and angjotensin II may influence the
From the Departments of Physiology (H.W., J.I., P.F.) and
Histology (EJ-)> University of Goteborg, Gdteborg, Sweden.
Supported by grants 00016, 02855, 07120, and 07685 from the
Swedish Medical Research Council, the Gothenburg Society of
Medicine, the Swedish Society of Medicine, the Royal Swedish
Society of Science, the Research Funds at Sahlgren's Hospital, and
the Faculty of Medicine, Gflteborg, Sweden.
Address for correspondence: Hakan WShlander, Department of
Physiology, University of Gdteborg, P.O. Box 33031, S-400 33
GOteborg, Sweden.
Received November 27, 1990; accepted in revised form Jury 23,
1991.
hypertrophic process.2-3 The rapidly developing left
ventricular hypertrophy after experimentally induced
renal artery stenosis (two-kidney, one clip [2K1C]
Goldblatt hypertension) has been suggested to constitute a local response to the increased pressure
load.4 However, in addition to the intermittent blood
pressure peaks that occur soon after renal artery
constriction,5 the level of circulating angiotensin II is
markedly elevated, which might contribute to the
development of left ventricular hypertrophy as may
also locally produced growth factors6 exercising autocrine or paracrine trophic effects. Moreover, interest has also been directed toward the role of protooncogenes as possible factors involved in regulation
of myocardial hypertrophy.7
Insulin-like growth factor I (IGF-I), which is identical to somatomedin-C, is a 70-amino acid peptide
that has a high degree of sequence homology to
insulin.8 The IGFs have traditionally been considered to be synthesized in the liver under growth
hormone regulation and released into the circulation
26
Hypertension
Vol 19, No 1 January 1992
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
to mediate the growth-promoting effects of growth
hormone.9 However, there is now considerable evidence showing synthesis of IGF-I in multiple extrahepatic tissues, including heart and skeletal muscle,10
and paracrine/autocrine mechanisms of action have
been proposed.
Studies using specific complementary DNA
(cDNA) probes for IGF-I have demonstrated the
presence of IGF-I messenger RNA (mRNA) in a
variety of rat tissues including heart and skeletal
muscle.1112 Expression of IGF-I mRNA in rat heart
was found to be partly regulated by growth hormone
with decreased levels after hypophysectomy, and
IGF-I mRNA levels were restored after growth hormone treatment.1112 Moreover, increased expression
of IGF-I mRNA has been demonstrated in tissues
regenerating after injury, both in intact and hypophysectomized rats,13 suggesting that IGF-I mRNA
expression may be independent of growth hormone
under certain conditions.
It was recently shown by Engelmann et al14 that
ventricular RNA from neonatal normotensive and
spontaneously hypertensive rats contained IGF-I
mRNA in low abundance with a relative higher
abundance in the hypertensive rats. In addition, in
situ hybridization revealed IGF-I mRNA expression
localized in myocytes and occasionally in endothelial
cells.
To elucidate a possible functional role of IGF-I as
a possible "second messenger" or permissive factor
during developing cardiac hypertrophy, 2K1C hypertension was induced in adult male rats. Abundance of
IGF-I mRNA was quantified using a solution hybridization assay, and the distribution of the IGF-I
protein across the myocardial wall was visualized by
immunohistochemistry.
Methods
General Procedures
Goldblatt or 2K1C hypertension was induced in
male normotensive Wistar rats (Mollegaard Breeding
Center Ltd., Ejby, Denmark), weighing about 180 g,
by placing a silver clip (diameter, 0.18 mm) around
the left renal artery leaving the right kidney intact. A
sham operation merely exposing the left renal artery
was performed in control rats. Anesthesia was induced by the short-lasting barbiturate methohexital
sodium (Brevital; Eli Lilly and Co., Indianapolis,
Ind.) (75 mg • kg"1 body wt i.p.). Ninety-seven percent (60 of 62) of the rats survived surgery, and no
other deaths occurred during the course of the study.
Experiments were then performed on groups of 10
2K1C and 10 sham-operated rats 2, 4, or 7 days
postoperatively.
On the day of experimentation, rats were weighed,
and their systolic blood pressure and heart rate were
measured by tail plethysmography (Narco BioSystems, Houston, Tex.). The animals were then anesthetized by methohexital sodium, and the hearts were
excised. The atria and great vessels were trimmed
away, and the ventricles were separated, blotted, and
weighed before being frozen in liquid nitrogen (N2)
and then stored at -20°C until further analysis could
be performed. The time from excision to freezing
never exceeded 5 minutes.
Preparation of Total Nucleic Acid
The tissue was homogenized and digested with
proteinase-K added to a sodium dodecyl sulfatecontaining buffer, as previously described.15 A subsequent extraction with phenolchloroform was then
performed.15
Solution Hybridization Assay
Quantitation of IGF-I mRNA was achieved using
an RNA-probe labeled with [35S]uridine-triphosphate, as described in detail previously.15-16 The
radioactive probe and the unlabeled synthetic IGF-I
mRNA strand were prepared according to the
method of Melton et al.17 In brief, the DNA clone
used is a 153-base pair genomic subclone (In pSP 64
in both orientations) of mouse IGF-I corresponding
to exon 3 (by analogy to human IGF-I). Studies by
Bell et al18 and Shimatsu and Rotwein19 suggest that
two forms of IGF-I mRNA exist in both mouse and
rat: IGF-IA mRNA and IGF-IB mRNA. The structure of the probe used in the present study corresponds to a part of the C-peptide, the whole A- and
D-peptide, and the part of the E-peptide that is
identical in both the 1A and IB prepro-IGF-I molecules. Therefore, the probe permitted the detection
of both the 1A and IB forms of IGF-I mRNA. The
mouse IGF-I complementary RNA (cRNA) probe
was hybridized at 70°C to total nucleic acid (TNA)
samples essentially as described.15 Incubations were
performed in microcentrifuge vials (Eppendorf) in a
volume of 40 /il containing 0.6 mol NaCl/1, 30 mmol
Tris-HO/1 (pH 7.5), 4 mmol EDTA/1, 0.1% sodium
dodecyl sulfate, 10 mmol dithiothreitol/1, and 25%
(vol/vol) formamide. After overnight incubation, the
samples were treated with RNase for 45 minutes at
37°C by adding 1.0 ml of a solution containing 40 /ig
RNase A and 2 /ig RNase T! (Boehringer-Mannheim, Mannheim, FRG) and 100 /ug of herring sperm
DNA (Sigma Chemical Co., St. Louis, Mo.) to each
sample. The radioactivity protected from RNase digestion by hybridization to mRNA was precipitated
by the addition of 100 fil trichloroacetic acid (6
mol/1) and collected on glass fiber filter (Whatman
GF/C; Whatman Ltd, Maidstone, UK). The hybridization signal of each sample was compared with a
standard curve constructed from incubations with
known amounts of IGF-I mRNA in a standard tissue
TNA preparation. This standard TNA preparation
was originally compared with the synthetic nucleotide mRNA strand.20 Each TNA sample was analyzed in triplicate. The DNA content was assayed
according to Labarca and Paigen,21 and results are
expressed as IGF-I mRNA/DNA (lO"18 mol//ig).
In a separate experiment, 0-actin mRNA was
analyzed in the left and right ventricles at 4 days
Wahlander et al Insulin-like Growth Factor in Hypertension
2
27
130-
I
O
O2K1C
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
FIGURE 2. Line plot shows effect of two-kidney, one clip
(2K1C) procedure on blood pressure. Male normotensive
Wistar rats weighing approximately 180 g were equipped with a
silver clip around the left renal artery leaving the contralateral
kidney intact. In control rats, a sham operation was performed
merely exposing the left renal artery. On the day of experimentation 2, 4, or 7 days after operation, systolic blood pressure
was measured by tail plethysmography. Blood pressure levels
are expressed as millimeters of mercury. Values are
mean±SEM. *p<0.05; **p<0.01.
FIGURE 1. Photomicrographs show serial sections from the
left ventricle 4 days after induction of renal hypertension with
a clip stenosing the left renal artery. Panel a: Incubation with
antiserum. Panel b: Incubation with antiserum preadsorbed
with synthetic insulin-like growth factor I. Bar, 50 pm.
after induction of renal hypertension or performance of sham operation using the same protocol
as described above. The probe used was a 1.9 kb
pair fragment of rat ^-actin cloned into a pGEM
vector (Promega, Madison, Wis.).
Immunohistochemistry
Hearts were rapidly removed and frozen in liquid
nitrogen. Transverse cryostat sections, 6-^xn thick,
were prepared from the right and left ventricle of
each heart. The sections were fixed in methanol at
-20°C and were then transferred to phosphatebuffered saline. After preincubation with 5% fat free
milk in phosphate-buffered saline, the sections were
incubated at 4°C overnight with a polyclonal antiserum raised in rabbits against human IGF-I (K1792;
kindly supplied by KabiGen AB, Stockholm, Sweden;
described by Nilsson et al22) and then with donkey
anti-rabbit immunoglobulin, peroxidase-linked
F(ab')2 fragment (Amersham, little Chalfont, UK)
for 1 hour at room temperature. The reaction was
visualized by the diaminobenzidine reaction.
Control sections incubated without the primary
antiserum showed unspecific staining of scattered
eiythrocytes but were otherwise negative. In sections
incubated with the primary antiserum preadsorbed
with purified IGF-I, a significant reduction in staining intensity was seen (Figure 1).
Statistical Methods
Statistical methods used were two-way analysis of
variance, followed by Student-Newman-Keuls multiple test between individual groups.
Results
To elucidate a possible role of IGF-I during development of cardiac hypertrophy, 2K1C hypertension
was produced in male Wistar rats. Postoperative
body weight developed in a similar manner in 2K1C
and sham-operated animals (180±2.2 g versus
183±1.9 g, 199±3.0 g versus 209±2.0 g, and 217±1.0
g versus 213±1.5 g at 2, 4, and 7 days, respectively).
Two days after surgery blood pressure was significantly elevated in clipped rats compared with shamoperated controls (Figure 2). The elevated blood
pressure persisted throughout the 7-day period of
study. Left ventricular weight remained largely unchanged 2 days after clipping but increased on the
fourth and seventh day after clipping, whereas no
change in right ventricular weight was evident at any
time point (Figures 3 and 4).
Quantitation of IGF-I mRNA, achieved using a
solution hybridization assay, revealed a fivefold increase of left ventricular levels of IGF-I mRNA 4 days
after clipping compared with sham-operated controls
(Figure 3). This difference was, however, not evident
after 7 days of renal artery constriction. In addition, as
shown in Figure 4, the right ventricular IGF-I mRNA
content was unaltered in renal hypertensive rats when
compared with normotensive control rats.
To investigate whether the increased levels of
IGF-I mRNA were due to a specific activation of the
IGF-I gene or part of a generalized increase in RNA,
levels of /3-actin mRNA were determined in the left
28
Hypertension
Vol 19, No 1 January 1992
.5-
•+-.
I
•
I Sh am
rh
2K1C
. 10-
2 days
Left
4 days
7 days
2 days
•3-1
ventricle
Right
4 days
7 days
ventricl
I 1
1 I
2K1C
.2-
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
5*
1-
V
2 days
4 days
2 days
7 days
4 days
7 days
FIGURE 3. Bar graphs show effect of two-kidney, one clip
(2K1C) hypertension on left ventricular weight (upper panel)
and insulin-like growth factor I (1GF-I) messenger RNA
(mRNA) content (lower panel). Male normotensive Wistar
rats weighing approximately 180 g were equipped with a silver
clip around the left renal artery leaving the contralateral kidney
intact. In control rats, a sham operation was performed merely
exposing the left renal artery. Experiments were then performed
2, 4, or 7 days after operation when the rats were weighed, and
hearts were excised and processed (see "Methods"). Left
ventricular weight is expressed as grams and levels of IGF-I
mRNA are expressed as 10~'s moles per microgram DNA.
Values are mean±SEM. **p<0.01.
FIGURE 4. Bar graphs show effect of two-kidney, one clip
(2K1C) hypertension on right ventricular weight (upper
panel) and insulin-like growth factor I (IGF-I) messenger
RNA (mRNA) content (lower panel). Male normotensive
Wistar rats weighing approximately 180 g were equipped with a
silver clip around the left renal artery leaving the contralateral
kidney intact In control rats, a sham operation was performed
merely exposing the left renal artery. Experiments were then
performed 2, 4, or 7 days after operation when the animals
were weighed, and hearts were excised and processed (see
"Methods"). Right ventricular weight is expressed as grams
and levels of IGF-I mRNA are expressed as 10'" moles per
microgram DNA. Values are mean±SEM.
and right ventricle of 2K1C and sham-operated rats 4
days after surgery. Table 1 shows that there was no
difference between the levels of 0-actin mRNA in
2K1C and sham-operated rats.
In both the left and right ventricles from shamoperated animals, scattered myocytes showed cytoplasmatic staining for IGF-I. Some unspecific staining of the connective tissue was usually observed
(Figure 5A). Four days after clipping, an increased
number of myocytes showed staining for IGF-I in the
left ventricle (Figures 5B and 5C), whereas only a few
myocytes were stained in the right ventricle (Figure
5D). This pattern remained consistent to the seventh
day after induction of renal hypertension (Figures 5E
and 5F). In the left ventricle, single cells or groups of
cells showed distinct staining, whereas other cells in
the immediate vicinity were negative (Figure 5F). It
was also possible to discriminate between the myo-
cardial wall layers, and here the number of IGF-I
positive cells as markedly higher in the subendocardial part of the heart wall, while fewer positively
TABLE 1. Effect of Two-Kidney, One Clip Hypertension on
Left and Right Ventricular £-Actln mRNA Content 4 Days
After Operation
Control values (%)
Left ventricle
Sham
100
2K1C
102
Right ventricle
Sham
100
2K1C
101
Quantities of 0-actin mRNA are expressed as a percentage of the
control values (sham-operated rats=100%). 2K1C, two-kidney, one
dip.
W&hlander et al
Insulin-like Growth Factor in Hypertension
29
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
FIGURE 5. Photomicrographs show cryostat sections from rat hearts processed to demonstrate insulin-like growth factor I (IGF-I)
immunoreactivity by indirect immunohistochemistry. Panel A: Section from the left ventricle of a sham-operated animal No
staining is seen in myocytes. Connective tissue shows unspedfic staining. Panels B and C: Section from the left ventricle 4 days after
induction of renal hypertension showing myocytes in cross (panel B) and longitudinal (panel C) sections. There is a focal expression
of IGF-I immunoreactivity showing distinctly IGF-I positive cells (arrow) mingled with negative cells (arrow heads). Panel D:
Section from the right ventricle of the same animal as in panel B. There is no staining of myocytes. Panel E: Section from the left
ventricle 7 days after the operation. Low magnification showing IGF-I positive myocytes mainly in the subendocardial region (left,
arrows). Panel F: Higher magnification of panel E shows sharp demarcation between unstained and IGF-I positive myocytes
(arrows). Peroxidase/DAB. Bars, 100
30
Hypertension
Vol 19, No 1 January 1992
stained cells were observed in the subepicardial
region (Figure 5E).
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
Discussion
The main findings in this study are that early renal
hypertension in Wistar rats results in increased levels
of IGF-I mRNA and IGF-I protein in the left but not
in the right ventricle. An elevated resting blood
pressure was found as soon as the second day after
renal artery constriction, whereas expression of
IGF-I mRNA was still unchanged from control rats
at this time. The increase in IGF-I mRNA peaked
after 4 days and then declined, and the pattern of
IGF-I protein was similar at 4 and 7 days after renal
artery constriction.
In recent years, an increasing interest has been
focused on trophic factors or molecules acting as
modulators of cardiovascular structural adaptation in
various forms of hemodynamic overload. There are a
vast number of potential endogenous molecules in
the myocardium that could exercise a hypertrophic
influence. Hammond et al6 found that protein synthesis in Langendorff-perfused hearts was stimulated
when homogenate from hearts of dogs with aortic
constriction was added to the perfusate. A similar
stimulating effect in isolated neonatal cardiomyocytes has been shown for hearts of adult spontaneously hypertensive rats.23 This group also purified the
stimulatory factor and found it to be a protein with a
molecular weight of 8,500 d. It is tempting to speculate that this protein in fact might be IGF-I, which
has been shown to have a molecular weight of 7,650
d.24 Speaking in favor of this hypothesis is the finding
that IGF-I mRNA is more abundant in hearts from
3-day-old spontaneously hypertensive rats than in
normotensive Wistar-Kyoto controls.14 Moreover, it
has previously been demonstrated with immunohistochemistry that expression of IGF-I is transiently
increased in activated satellite cells during regeneration after toxic or ischemic injury.25 Furthermore,
extracted IGF-I mRNA levels measured with solution hybridization showed a 20-fold increase during
the regeneration process.13 High levels of IGF-I
mRNA have also been seen in hypophysectomized
rats after injury, and similar results were obtained in
aortic smooth muscle cells after balloon denudation.26 These latter findings indicate that IGF-I
expression, under some conditions, is independent of
growth hormone and under such conditions autocrine/paracrine mechanisms for IGF-I regulation and
IGF-I action have been proposed.
The stimulus for the increase in left ventricular
IGF-I observed in the present study is not clear.
However, the fact that the increase of both IGF-I
mRNA and protein was seen solely in the left and not
in the right ventricle directs attention toward a
stimulus that would affect the left ventricle only. This
implies that mechanical factors would be likely to be
involved. Increased coronary perfusion pressure
stimulates protein synthesis in isolated perfused
hearts27 as well as external load in the absence of
other trophic factors in isolated feline cardiomyocytes28 and the mere presence of contractions in
cultured neonatal cardiomyocytes.29 However, coronary perfusion pressure and flow as well as contractions per se will also affect both ventricles to the same
extent and are thus less probable as stimuli to IGF-I
synthesis. The increased left ventricular work load
would be a more likely candidate to explain the
raised IGF-I levels in the left ventricle, and this
assumption is also strengthened by the present observation that IGF-I protein seems to be more abundant in the inner layers of the myocardium (Figure
5E). In these layers tension, as well as wall stress, is
high and declines toward the epicardial surface.30
Wall stress may thus play an important role as
stimulus for IGF-I synthesis under the present conditions. This may also explain why no elevation of
right ventricular IGF-I levels was found; renal hypertension is not associated with an increased pressure
in the pulmonary circulation, and consequently a
normal wall tension is expected in the right ventricle.
This hypothesis is further substantiated by the recent
finding that hydrostatic pressure can increase secretion of IGF-I from cultured endothelial cells.31 Moreover, increasing tension (active as well as passive) has
been shown to increase protein synthesis in isolated
papillary muscle through an increase in Na+ influx,
probably through fast stretch-activated cation channels,32 and IGFs can act synergistically with monovalent ion influx to increase DNA synthesis.33 Thus,
from the present in vivo results, one can suggest a
possible link between increased tension/wall stress,
increased myocardial IGF-I synthesis, and a hypertrophic response in the myocardium, at least in this
early phase of hypertension. However, direct experiments to establish the existence of such a link still
remain to be performed.
Cardiac hypertrophy and possibly also myocardial
IGF-I production may be modified by endocrine or
neural factors with trophic properties. Growth hormone is obviously important for regulation of IGF-I
synthesis, but as pointed out above, growth hormone-independent IGF-I synthesis has been described under various conditions. In the present
study, a change in growth hormone levels would
affect both the left and the right ventricle equally,
and it is therefore not likely that growth hormone is
the sole mediator of the increased IGF-I synthesis.
Nevertheless, a normal growth hormone level may
well be required to elicit the present IGF-I response
since it seems to be a permissive factor for the
development of left ventricular hypertrophy after
aortic constriction and renal artery stenosis.34-35 Catecholamines such as epinephrine or norepinephrine
have been shown to induce hypertrophy of cultured
neonatal cardiomyocytes via an a,-receptor-dependent increase in protein synthesis.2 Similar findings
have been reported in both isolated perfused working
hearts and cultured myocytes from adult rats.36 Yet,
in another study, cardiac sympathetic denervation
Wihlander et al
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
did not influence right ventricular hypertrophy after
banding of the pulmonary artery.37 Thus, the discrepancy between in vitro and in vivo results regarding
the effects of catecholamines makes their role in the
development of cardiac hypertrophy unresolved. In
the present model, sympathetic outflow to the heart
may increase as a consequence of elevated levels of
circulating angiotensin II, due to either enhanced
presynaptic release of norepinephrine or facilitation
of the effects of norepinephrine postjunctionalry.
This would most likely affect both ventricles in a
similar manner, and thus, catecholamines would not
by themselves explain the effect on left ventricular
IGF-I synthesis. Angiotensin II represents yet another hormone that may be linked to development of
myocardial hypertrophy. The circulating levels of
angiotensin II increase in early renal hypertension,
and in addition, angiotensin II has been shown to
increase cardiac contractility and contractile frequency in cultured myocytes.38 Moreover, angiotensin II possesses hypertrophic properties in cultured
neonatal chick heart cells,3 but in myocytes from
adult rats, no stimulatory effect on protein synthesis
could be detected in response to angiotensin II.36
This discrepancy may be attributed to the fact that
angiotensin II receptors in adult rat hearts are mainly
confined to the conduction system and are virtually
absent in ventricular myocytes.39 Thus, the role of
circulating angiotensin II in the development of
cardiac hypertrophy is still unclear, and as pointed
out above, it does not seem to be the most probable
candidate as stimulus for the increased left ventricular IGF-I synthesis observed in the present study.
Angiotensin II may also be produced locally in the
myocardium, and it has recently been shown that
angiotensin II is required for the development of left
ventricular hypertrophy after aortic constriction,
mimicking the situation for growth hormone.40-41
Furthermore, an increase in myocardial angiotensin
converting enzyme has been demonstrated in rats
with aortic stenosis,42 possibly generating higher tissue levels of angiotensin II than circulating concentrations would imply. Thus, although humoral or
nervous stimuli by themselves do not seem to be able
to induce the increased levels of left ventricular IGF-I,
any such factor may be important through interactions
with mechanical or hemodynamic stimuli.
In recent years an increasing number of investigations have emphasized the possible role of locally
produced growth factors in the early stages of cardiovascular hypertrophy, and IGF-I is, definitely, not
the only trophic molecule produced by the myocardium. The proto-oncogenes c-myc, c-fos, and c-Haras increased in the myocardium in response to
mechanical stimuli as well as vasoactive agents such
as angiotensin II, vasopressin, and phenylephrine.43-45 Furthermore c-sis, which codes for the B
chain of platelet-derived growth factor, is present in
the myocardium and can also induce expression of
c-myc and c-fos. Any of these factors could theoretically have influenced or modulated the production of
Insulin-like Growth Factor in Hypertension
31
IGF-I in the 2K1C model of hypertension. However,
none of these studies have been able to show a
definite functional role of the proto-oncogenes in the
hypertrophic process. Furthermore, in most cases
only the genetic precursor and not the actual peptide
was measured, and the possibility exists that the
genes were transcribed without translation into the
peptide end product.
In conclusion, expression of IGF-I mRNA and
synthesis of IGF-I protein were enhanced in the left
but not in the right ventricle 4 days after renal artery
constriction. The enhancement was detected after an
established increase in blood pressure could be demonstrated. The augmentation of IGF-I synthesis
seems to be coupled to the increased left ventricular
work load and not to circulating humoral factors such
as growth hormone or angiotensin II, thus suggesting
an autocrine/paracrine regulation of IGF-I under
these conditions. Because of the finding of more
IGF-I protein in the endocardial than in the epicardial layers, wall stress seems to be the most likely
candidate as mechanical stimulus for IGF-I synthesis.
Thus, the present study demonstrates the ability of
the myocardium itself to produce a molecule with
possible trophic properties in direct response to a
mechanical stimulus.
Acknowledgments
The gift of antiserum against IGF-I from KabiGen
AB, Stockholm, Sweden, is gratefully acknowledged.
The excellent technical assistance of Gunnel Andersson is greatly appreciated. We thank Margareta
Nordlander for fruitful discussions.
References
1. Fribcrg P: Structural and functional adaptation in the rat
myocardium and coronary vascular bed caused by changes in
pressure and volume load. Acta Physiol Scand 1985;124(suppl
540):l-47
2. Simpson P: Stimulation of hypertrophy of cultured neonatal
rat heart cells through an o^-adrenergic receptor and induction of beating through an or,- and /3,-adrenergic receptor
interaction. Ore Res 1985^6:884-894
3. Aceto JF, Baker KM: [Sar,]angiotensin II receptor-mediated
stimulation of protein synthesis in chick heart cells. Am J
Physiol 1990;258:H806-H813
4. Lundgren Y, Hallback M, Weiss L, Folkow B: Rate and extent
of adaptive cardiovascular changes in rats during experimental
renal hypertension. Ada Physiol Scand 1974;91:103-115
5. Helmchen U, Bohle RM, Kneissler U, Groene H: Intrarenal
arteries in rats with early two-kidney one-dip hypertension.
Hypertension 1984;6(suppl ni):III-87-III-92
6. Hammond GL, Lai YK, Markert CL: The molecules that
initiate cardiac hypertrophy are not species-specific. Science
1982;216:529-531
7. Simpson PC: Proto-oncogenes and cardiac hypertrophy. Anna
Rev Physiol 1988^1:189-202
8. Mapper DG, Svoboda ME, van Wyk JJ: Sequence analysis of
somatomedin-C: Confirmation of sequence identity with insulin-like growth factor I. Endocrinology 1983;112:2215-2217
9. Salmon WD Jr, Daughaday WH: A hormonaHy controlled
serum factor which stimulates sulfate incorporation by cartilage in vitro. J Lab din Med 1957;49:825-836
10. D'Ercole AJ, Stiles AD, Underwood LE: Tissue concentrations of somatomedin-C: Further evidence for multiple sites of
32
Hypertension
Vol 19, No 1 January 1992
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
synthesis and paracrine or autocrine mechanisms of action.
Proc Nad Acad Sci U S A 1984;81:935-939
11. Murphy LJ, Bell GI, Duckworth ML, Friesen HG: Identification, characterization and regulation of a rat complementary
deoxyribonudeic acid which encodes insulin-like growth factor
I. Endocrinology 1987;121:684-691
12. Isgaard J, Nilsson A, Vikman K, Isaksson OGP: Growth
hormone regulates the level of insulin-like growth factor I in
rat skeletal muscle. / Endocrinol 1989;120:107-112
13. Edwall D, Schalling M, Jennische E, Norstedt G: Induction of
insulin-like growth factor I messenger ribonucleic acid during
regeneration of rat skeletal muscle. Endocrinology 1989;124:
820-825
14. Engelmann GL, Boehm KD, Haskell JF, Khairallah PA, Ilan
J: Insulin-like growth factors and neonatal cardiomyocyte
development: Ventricular gene expression and membrane
receptor variations in normotensive and hypertensive rats. Mol
Cell Endocrinol 1989;63:1-14
15. Durnam DM, Palmiter RD: A practical approach for quantitating specific mRNAs by solution hybridization. Anal Biochem 1983;131 J85-393
16. Mathews LS, Norstedt G, Palmiter RD: Regulation of insulinlike growth factor I gene expression by growth hormone. Proc
Nad Acad Sci USA 1986;83:9343-9347
17. Melton DA, Krieg A, Rebagliati MR, Maniatis T, Zinn K,
Green MR: Efficient in vitro synthesis of biologically active
RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 1984;12:
7035-7056
18. Bell GI, Stempien MM, Fong NM, Rail LB: Sequences of liver
cDNAs encoding two different mouse insulin-like growth
factor I precursors. Nucleic Acids Res 1986;14:7873-7882
19. Shimatsu A, Rotwein P: Mosaic evolution of the insulin-like
growth factors: Organization, sequence and expression of rat
insulin-like growth factor I gene. / Biol Chan 1987;262:
7894-7900
20. Isgaard J, MSIIer C, Isaksson OGP, Nilsson A, Mathews LS,
Norstedt G: Regulation of insulin-like growth factor messenger ribonucleic acid in rat growth plate by growth hormone.
Endocrinology 1988;122:1515-1520
21. Labarca C, Paigen K: A simple, rapid and sensitive DNA assay
procedure. Anal Biochem 1980;102:344-352
22. Nilsson A, Isgaard J, Lindahl A, DahlstrOm A, Skottner A,
Isaksson OGP: Regulation by growth hormone of number of
chondrocytes containing IGF-I in rat growth plate. Science
1986;233:571-574
23. Sen S, Petscher C, Ratliff N: A factor that initiates myocardial
hypertrophy in hypertension. Hypertension 1987:9:261-267
24. Rinderknecht E, Humbel RE: The amino acid sequence of
human insulin-like growth factor I and its structural homology
with proinsulin. J Biol Chan 1978;253:2769-2776
25. Jennische E, Skottner A, Hansson H-A; Satellite cells express
the trophic factor IGF-I in regenerating skeletal muscle. Acta
Physiol Scand 1987;1293-15
26. Cercek B, Fishbein MC, ForTester JS, Helfant RH, Fagin JA:
Induction of insulin-like growth factor I messenger RNA in rat
aorta after balloon denudation, drc Res 1990;66:l 755-1760
27. Takala T: Protein synthesis in the isolated perfused rat heart.
Basic Res Cordial 1981;76:44-61
28. Mann DL, Kent RL, Cooper G IV: Load regulation of the
properties of adult feline cardiocytes: Growth induction by
cellular deformation. Circ Res 1989;64:1079-1090
29. Me Derroott PJ, Morgan HE: Contraction modulates the
capacity for protein synthesis during growth of neonatal heart
cells in culture. Circ Res 1989;64:542-553
30. Mirsky I: Elastic properties of the myocardium: A quantitative
approach with physiological and clinical applications, in Berne
RM, Sperelakis N, Geiger SR (eds): Handbook of Physiology:
The Cardiovascular System, Volume I. Washington, DC, American Physiological Society, 1979, pp 497-532
31. Taylor WR, Delafontaine P, Griendling KK, Nerem RM,
Alexander RW: Pressure induces insulin-like growth factor-1
secretion by endothelial cells (abstract). Hypertension 1990;16:
335
32. Kent RL, Hoober JK, Cooper G IV: Load responsiveness of
protein synthesis in adult mammalian myocardium: Role of
cardiac deformation linked to sodium influx. Circ Res 1989;64:
74-85
33. Rozengurt E, Ober SS: The role of early signaling events in the
mitogenic response. News Physiol Sci 1990^:21-24
34. Beznak M: The restoration of cardiac hypertrophy and blood
pressure in hypophysectomized rats by large doses of lyophilized anterior pituitary and growth hormone. J Physiol
1954;124:64-74
35. Folkow B, Isaksson OPG, Karlstrom G, Lever AF, Nordlander
M: The importance of hypophyseal hormones for structural
cardiovascular adaptation in hypertension. / Hypertens 1988;
6(suppl 4):S166-S169
36. Fuller SJ, Gaitanaki CJ, Sugden PH: Effects of catecholamines
on protein synthesis in cardiac myocytes and perfused hearts
isolated from adult rats. Biochem J 1990;266:727-736
37. Cooper G IV, Kent RL, Uboh CE, Thompson EW, Marino
TA: Hemodynamic versus adrenergic control of cat right
ventricular hypertrophy. J Clin Invest 1985;74:1403-1414
38. Allen IS, Cohen NM, Dhallan RS, Gaa ST, Lederer WJ,
Rogers TB: Angiotensin II increases spontaneous contractile
frequency and stimulates calcium current in cultured neonatal
rat heart myocytes: Insights into the underlying biochemical
mechanisms. Circ Res 1988;62:524-534
39. Saito K, Gutkind JS, Saavedra JM: Angiotensin II binding
sites in the conduction system of rat hearts. Am J Physiol
1987;253:H1618-H1622
40. Linz W, Sch6lkens BA, Ganten D: Converting enzyme inhibition specifically prevents the development and induces regression of cardiac hypertrophy in rats. Clin Exp Hypertens [A]
1989;ll:1325-1350
41. Baker KM, Chernin MI, Wixson SK, Aceto JF: Reninangiotensin system involvement in pressure-overload cardiac
hypertrophy in rats. Am J Physiol 1990;259:H324-H332
42. Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS,
Lorell BH: Increased rat cardiac angiotensin converting
enzyme activity and mRNA expression in pressure overload
left ventricular hypertrophy. / Clin Invest 1990;86:1913-1920
43. Bauters C, Moalic JM, Bercovici J, Mouas C, Emanoil-Ravier
R, Schiaffino S, Swyngedauw B: Coronary flow as a determinant of c-myc and c-fos proto-oncogene expression in an
isolated adult rat heart. / Mol Cell Cardiol 1988;20:97-101
44. Komuro I, Kurabayashi M, Takaku F, Yazaki Y: Expression of
cellular oncogenes in the myocardium during the developmental stage and pressure-overloaded hypertrophy of the rat
heart. On: Res 1988;62:1075-1079
45. Moalic JM, Bauters C, Himbert D, Bercovici J, Mouas C,
Guicheney P, Baudoin-Legros M, Rappaport L, EmanoilRavier R, Mezger V, Swynghedauw B: Phenylephrine, vasopressin and angiotensin II as determinants of proto-oncogene
and heat-shock protein gene expression in adult rat hearts and
aorta. J Hypertens 1989;7:195-201
KEY WORDS • rat studies • insulin-like growth factor
renal hypertension • hypertrophy • wall stress
•
Left ventricular insulin-like growth factor I increases in early renal hypertension.
H Wåhlander, J Isgaard, E Jennische and P Friberg
Hypertension. 1992;19:25-32
doi: 10.1161/01.HYP.19.1.25
Downloaded from http://hyper.ahajournals.org/ by guest on May 2, 2017
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1992 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://hyper.ahajournals.org/content/19/1/25
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally
published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not
the Editorial Office. Once the online version of the published article for which permission is being requested
is located, click Request Permissions in the middle column of the Web page under Services. Further
information about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/