Download Differential expression of TNF-, IL-6, and IGF

Am J Physiol Heart Circ Physiol 282: H926–H934, 2002.
First published November 8, 2001; 10.1152/ajpheart.00436.2001.
Differential expression of TNF-␣, IL-6, and IGF-1
by graded mechanical stress in normal rat myocardium
Received 22 May 2001; accepted in final form 5 November 2001
Palmieri, Emiliano A., Giulio Benincasa, Francesca
Di Rella, Cosma Casaburi, Maria G. Monti, Giuseppe
De Simone, Lorenzo Chiariotti, Lucio Palombini, Carmelo B. Bruni, Luigi Saccà, and Antonio Cittadini.
Differential expression of TNF-␣, IL-6, and IGF-1 by graded
mechanical stress in normal rat myocardium. Am J Physiol
Heart Circ Physiol 282: H926–H934, 2002. First published
November 8, 2001; 10.1152/ajpheart.00436.2001.—An isovolumic normal rat heart Langendorff model was used to
examine the effects of moderate (15 mmHg) and severe (35
mmHg) mechanical stretch on the time course (from 0 to 60
min) of myocardial expression of tumor necrosis factor
(TNF)-␣, interleukin (IL)-6, and insulin-like growth factor
(IGF)-1 and their cognate receptors. After 10 min of moderate
stretch, TNF-␣ was de novo expressed, whereas constitutive
IL-6 and IGF-1 levels were slightly upregulated; no further
changes occurred up to 60 min. In comparison, severe stretch
resulted in a higher and progressive increase in TNF-␣, IL-6,
and IGF-1 expression up to 20 min. After 20 min, whereas
TNF-␣ expression further increased, IL-6 and IGF-1 levels
progressively reduced to values lower than those observed
under moderate stretch and in unstretched (5 mmHg) control
myocardium (IL-6). Mechanical stretch did not significantly
alter the expression of the cognate receptors. Indeed, the
TNF-␣ receptor (p55) tended to be progressively upregulated
under severe stretch over time. The current data provide the
first demonstration that TNF-␣, IL-6, and IGF-1 ligandreceptor systems are differentially expressed within the normal rat myocardium in response to graded mechanical
stretch. Such findings may have potential implications with
regard to compensatory hypertrophy and failure.
heart; hemodynamic overload; gene expression
occurring immediately after myocardial infarction plays a critical
role in determining whether the noninfarcted cardiac
muscle will develop functionally adaptive hypertrophy
or eventually undergo decompensation and failure
THE MAGNITUDE OF HEMODYNAMIC OVERLOAD
Address for reprint requests and other correspondence: A. Cittadini, III Divisione di Medicina Interna, Via S. Pansini 5, 80131
Naples, Italy (E-mail: [email protected]).
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(28, 31). Although the extant literature indicates
that hemodynamic overload affects the myocardium
by mechanical stretch primarily (33), the biochemical mechanisms responsible for orchestrating these
phenotypically and prognostically different outcomes
remain unclear.
Accumulating evidence indicates that a portfolio of
endogenous autocrine/paracrine cytokines and growthpromoting factors are promptly synthesized within the
myocardium in response to mechanical stretch (14).
Although the role that these substances play is not
precisely defined, it has been proposed that they may
contribute to initiate and modulate critical responses
within the overloaded myocardium, such as myocyte
growth, apoptotic myocyte death, and reactive fibrosis,
which, in turn, are the main determining factors of the
final outcome of hemodynamic overload (5).
Among these endogenous molecules, increasing attention has been recently focused on tumor necrosis
factor (TNF)-␣, interleukin (IL)-6, and insulin-like
growth factor (IGF)-1. Independent investigators have
documented their early upregulation within the myocardium in experimental load-induced cardiac hypertrophy (9, 19, 30). Interestingly, although these peptides share the common ability to activate myocyte
growth (15, 18, 40), they exert different effects with
regard to apoptotic myocyte death and interstitial compartment. Specifically, whereas IL-6 and IGF-1 possess
univocal antiapoptotic properties (8, 38) and preserve
the integrity of the interstitial network (7, 15), TNF-␣
appears to serve a dual biological purpose. At “physiological” concentrations, it exerts cytoprotective effects
within the myocardium, including antioxidant and antiapoptotic effects (24, 26). In contrast, at “pathophysiological” concentrations, it stimulates myocyte apoptosis (22) and reactives myocardial fibrosis (23, 36).
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.
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society
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EMILIANO A. PALMIERI,1 GIULIO BENINCASA,2 FRANCESCA DI RELLA,1
COSMA CASABURI,1 MARIA G. MONTI,1 GIUSEPPE DE SIMONE,1 LORENZO CHIARIOTTI,4
LUCIO PALOMBINI,2 CARMELO B. BRUNI,3 LUIGI SACCÀ,1 AND ANTONIO CITTADINI1
1
Department of Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, and 2Department of
Scienze Biomorfologiche e Funzionali, Sezione di Anatomia Patologica e Citopatologia, and
3
Department of Biologia e Patologia Cellulare e Molecolare “L. Califano,” Facoltà di Medicina e
Chirurgia, Università degli Studi di Napoli “Federico II,” 80131 Naples; and 4Department of
Medicina Sperimentale e Clinica “G. Salvatore,” Facoltà di Medicina e Chirurgia,
Università degli Studi di Catanzaro “Magna Grecia,” 88100 Catanzaro, Italy
STRETCH-INDUCED MYOCARDIAL GROWTH FACTORS AND CYTOKINES EXPRESSION
Given this evidence, in the current study, we examined myocardial TNF-␣, IL-6, and IGF-1 expression,
and the levels of their cognate receptors, in response to
different degrees of acute mechanical stretch in the
adult rat. To this aim, we used an in vitro isolated,
isovolumic, buffer-perfused heart Langendorff preparation to achieve graded levels of mechanical stress by
inflating an intraventricular balloon at two different
levels of end-diastolic pressures. This model allowed us
to eliminate the confounding effects of circulating substances such as neurohormones, which are known to be
upregulated under in vivo hemodynamic overload.
METHODS
AJP-Heart Circ Physiol • VOL
loaded muscles by Blinks et al. (3) and modified for whole
heart studies by Kihara et al. (20).
Experimental protocol and signal recording. After 15–30
min at 25°C, the temperature was gradually increased to
37°C and kept constant by regulating the temperature of the
perfusate. After a 15-min stabilization period, the balloon
was further inflated to achieve an end-diastolic pressure of
⬃5 mmHg (unstretched control myocardium), 15 mmHg
(moderate stretch), or 35 mmHg (severe stretch). The coronary flow rate was adjusted to keep a constant tissue perfusion of 10 ml 䡠 min⫺1 䡠 g heart wt⫺1. After 10, 20, 40, and 60
min, respectively, the balloon was rapidly deflated to a volume just enough to obtain a pressure signal. After a 5-min
stabilization, perfusion was terminated, and the left ventricles, carefully separated from the right ventricles, were
quickly cross sectioned in two portions. One portion was
snap-frozen in liquid nitrogen for Northern and Western blot
analysis (see Northern blotting and Western blotting, respectively), and the other was formalin-fixed for immunohistochemistry (see Immunohistochemical analysis). Before, during, and after the course of experiments, the digital signals of
the left ventricular isovolumic pressure, aequorin light signals, and coronary perfusion pressure were simultaneously
recorded on a four-channel recorder and averaged in a computer (6), and the left ventricular pressure tracing was further analyzed using customized software (6) to obtain the
following parameters: peak left ventricular systolic pressure,
left ventricular end-diastolic pressure, left ventricular developed pressure, and maximum and minimum values of the
first pressure derivative with respect of time. At 10-min
intervals during experiments, a sample of the coronary venous effluent was collected in a calibrated cylinder over a
period of 1 min for measuring lactate production (Lactate
Reagent, Sigma) and lactate dehydrogenase release (Lactate
Dehydrogenase Reagent, Sigma).
Probe generation. Rat-specific cDNA probes for TNF-␣,
IL-6, IGF-1, and glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) were generated by sequential RT-PCR (GIBCOBRL Life Technologies) according to the manufacturer’s procedures. Total RNA from endotoxin-stimulated (10 ␮g/ml for
8 h) rat macrophage cells and the normal rat liver were used
as templates for the RT reactions. For the 540-bp TNF-␣
probe, the following oligonucleotides were used: sense primer
(5⬘-CGCTCTTCTGTCTACTGAAC-3⬘), corresponding to nucleotides 4,568–4,577; antisense primer (5⬘-TTCTCCAGCTGGAAGACTCC-3⬘),
corresponding
to
nucleotides
5,939–5,958 (GenBank Accession No. L00981). For the
650-bp IL-6 probe, the following oligonucleotides were used:
sense primer (5⬘-CTTCCCTACTTCACAAGTCC-3⬘), corresponding to nucleotides 3,206–3,225; antisense primer (5⬘GACCACAGTGAGGAATGTCC-3⬘), corresponding to nucleotides 7,187–7,206 (GenBank Accession No. M26745). For the
490-bp IGF-1 probe, the following oligonucleotides were used:
sense primer (5⬘-CATGTCGTCTTCACATCTCTTC-3⬘), corresponding to nucleotides 42–63; antisense primer (5⬘-GGCTCCTCCTACATTCTGTA-3⬘), corresponding to nucleotides
415–434 (GenBank Accession No. D00698). For the 428-bp
GAPDH probe, the following oligonucleotides were used:
sense primer (5⬘-CACCATCTTCCAGGAGCGAG-3⬘), corresponding to nucleotides 239–258; antisense primer (5⬘ACAGCCTTGGCAGCACCAGT-3⬘), corresponding to nucleotides 648–667 (GenBank Accession No. AF106860). All
specific amplified fragments were purified using QIAquick
Spin (QIAgen), labeled with [␣-32P]dATP and [␣-32P]dGTP
(Amersham Pharmacia Biotech) by a random priming procedure (10), and used as probes (see Northern blotting) at the
specific activity of at least 1 ⫻ 109 counts 䡠 min⫺1 䡠 ␮g⫺1.
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Adult male normal Wistar rats weighing 300–500 g (Stefano Morini, Reggio Emilia, Italy), fed with normal rat chow
and water ad libitum, were used for the whole heart experiments. All methods described conformed to the “Guiding
Principles for Research Involving Animals and Human Beings,” and the protocol was approved by the Animal Care
Committee of the University Federico II of Naples, Italy.
Isolated whole heart preparation. Rats were killed, and the
isolated hearts were placed in an isovolumic buffer-perfused
preparation according to the Langendorff technique, as previously described (6). Briefly, the rats were anesthetized by
an intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg), and 200 IU heparin were injected into the
femoral vein. One minute later, the hearts were quickly
excised and immersed in ice-cold Krebs-Henseleit solution
(see below), weighed, and mounted on a cannula inserted into
the ascending aorta. The hearts were retrogradely perfused
within 30 s after the thoracotomy using a Krebs-Henseleit
solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 KH2PO4,
1.5 CaCl2, 1.2 MgCl2, 23 NaHCO3, and 5.5 dextrose saturated with a 95% O2-5% CO2 gas mixture to a pH of 7.4. A
constant flow perfusion of 10 ml 䡠 min⫺1 䡠 g heart wt⫺1 was
achieved by means of a roller pump, and coronary perfusion
pressure was monitored by a Statham P23 Db transducer
(Gould; Cleveland, OH) connected to the perfusion line. This
value was chosen according to preliminary experiments with
graded ischemia that revealed an aerobic pattern of lactate
consumption measured according to Apstein et al. (1). Left
ventricular isovolumic pressure was measured by a second
Statham P23 DB transducer attached to a fluid-filled latex
balloon inserted into the left ventricle via the mitral valve.
Thebesian venous return from the left ventricle was emptied
via a drain inserted in parallel with the balloon. Cardiac
temperature was set at 25°C, measured by a temperature
probe inserted into the right ventricle, and the hearts were
paced at 3 Hz. The left intraventricular balloon was inflated
just enough to obtain a pressure signal to monitor preparation stability.
Aequorin loading. Aequorin loading, performed as previously described (6), was used to monitor qualitative and
quantitative change in intracellular calcium, which is a sensitive marker of myocardial ischemia (22). Briefly, 3–5 ␮l of
an aequorin-containing solution (1 ␮g/ml) were macroinjected into the interstitium of the inferoapical region of the
left ventricle. The heart was then positioned in a organ bath
with the aequorin-loaded area of the left ventricle directed
toward the cathode of a photomultiplier (model 9635QA,
Thorn-EMI, Gencom) and submerged in Krebs-Henseleit solution. The organ bath was enclosed in a light-occlusive
photographic bellows designed for studies with aequorin-
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STRETCH-INDUCED MYOCARDIAL GROWTH FACTORS AND CYTOKINES EXPRESSION
AJP-Heart Circ Physiol • VOL
sodium citrate buffer, pH 6.0, and endogenous peroxidase
activity was quenched with 0.3% H2O2 in 90% methanol.
Sections were incubated 1 h at 37°C with rat reactive affinity-purified polyclonal IgG specific for TNF-␣ (1:150), IL-6
(1:100), IGF-1 (1:100), TNF-R1 (1:50), IL-6-R␣ (1:25), and
IGF-1-R␣ (1:150) (Santa Cruz Biotechnology), followed by
30-min incubation at room temperature with the appropriated secondary biotinylated antibody (Santa Cruz Biotechnology). The presence of the specific protein was revealed by
incubating slides with streptavidin-horseradish peroxidase
complex (30 min) and then by adding diaminobenzidine
(DAB) chromogen as peroxidase substrate for 1 min (Dako
LSAB kit, Dako A/S). Specifically, the duration of incubation
with DAB was empirically chosen as the time resulting in the
optimal ratio of specific protein signal to unspecific staining
as determined by monitoring the peroxidase reaction under a
light microscope. Slides were weakly counterstained with
Harris’s hematoxylin and permanently mounted with a synthetic mounting medium. Control negative sections were
obtained for each left ventricular cross section by the same
procedure described above except for the omission of the
primary antibody incubation. All sections were examined
with a light microscope at ⫻250 and ⫻500 magnification.
Statistical analysis. The data are presented as means ⫾
SE of three independent experiments for each time and level
of stretch. One-way ANOVA followed by the Newman-Keuls
post hoc test was used for statistical comparisons of the
differences in aequorin light signals, lactate production, and
changes in developed pressure. Two-way ANOVA followed by
the Newman-Keuls post hoc test was used for statistical
comparisons of the differences in mRNA and protein expression for each of the candidate molecules at each time and
level of stretch. The threshold for statistical significance was
set at P ⬍ 0.05.
RESULTS
Compared with hearts perfused under control conditions (unstretched myocardium, 5 mmHg), no significant changes were detected during 60 min of moderate
(15 mmHg) and severe (35 mmHg) stretch in epicardial
diastolic [0.07 ⫾ 0.05, 0.09 ⫾ 0.02, and 0.08 ⫾ 0.03 nA,
respectively, P ⫽ not significant (NS)] and systolic
(0.40 ⫾ 0.09, 0.49 ⫾ 0.07, and 0.42 ⫾ 0.08 nA, respectively, P ⫽ NS) aequorin light signals or in lactate
production and lactate dehydrogenase release (data
not shown). Moreover, compared with unstretched
hearts, no significant changes were measured before or
after 60 min of moderate and severe stretch in developed pressure (percent difference: 2.86, 4.17, and
10.14, respectively, P ⫽ NS). Taken together, these
findings indicated that in our experimental settings
neither relevant ischemia nor mechanical tissue damage have occurred.
Figure 1 shows representative autoradiograms (A)
and corresponding densitometry (B) of total myocardial
RNA by Northern blotting for TNF-␣, IL-6, and IGF-1.
In unstretched/unperfused hearts (data not shown)
and throughout control perfusion (unstretched myocardium), TNF-␣ was undetectable, whereas both IL-6
(⬃1.5 kb) and IGF-1 (⬃7.5 kb) were constitutively and
stably expressed. Induction of moderate stretch was
accompanied by de novo TNF-␣ (⬃1.7 kb) expression
and significant upregulation of both IL-6 and IGF-1
levels, which appeared maximal after 10 min and re-
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Northern blotting. Total RNA was extracted from the
homogenized left ventricular myocardium using TRIzol
Reagent (GIBCO-BRL Life Technologies) according to the
manufacturer’s procedures. Northern blots (Hybond-N⫹,
Amersham Pharmacia Biotech) were performed using 1.2%
agarose gel electrophoresis under denaturing conditions according to standard procedures (35). Hybridizations were
carried out at 65°C in Rapid-hyb buffer (Amersham Pharmacia Biotech) with the specific cDNA probes, followed by washing at various final stringencies until radioactive background
was negligible, according to the manufacturer’s procedures.
Specifically, blots were sequentially hybridized, stripped, and
reprobed with the TNF-␣, IL-6, and IGF-1 probes and finally,
to correct for potential differences in the amount of RNA
loaded and transferred, with the cDNA probe for GAPDH.
Thus exposure time for each hybridization (⫺70°C with intensifying screens) was chosen within the linear response
range of the radiographic film (Kodak XAR), and quantitative
evaluation was approached by normalizing the scanning densitometric intensity of the specific autoradiograms for TNF-␣,
IL-6, and IGF-1 to the hybridization signal obtained with
GAPDH from the same lane (imaging densitometer model
GT-670, Bio-Rad). The sizes of the hybridized messengers
were estimated using the 28S and the 18S rRNA bands as
standards. Total RNAs from endotoxin-stimulated (10 ␮g/ml
for 8 h) rat macrophage cells and the normal rat liver were
used as positive controls for the TNF-␣, IL-6, and IGF-1
cDNA probes, respectively.
Western blotting. Western blotting experiments were performed according to standard procedures (13). Briefly, the
powdered left ventricular myocardium was homogenized in
JS lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1%
glycerol, 1% Triton X-100, 1.5 mM MgCl2, and 5 mM EGTA]
containing 1 mM phenylmethylsulfonyl fluoride and protease
inhibitor cocktail (complete Protease Inhibitors Cocktail Tablets, Roche Molecular Biochemicals). Lysates were clarified
by centrifugation at 10,000 g, and protein concentration was
estimated by a modified Bradford assay (Bio-Rad). Western
blots (Protran, Schleichen and Schuell) were performed using
15% SDS-PAGE under reducing conditions. High-range
Rainbow molecular weight markers (Amersham Pharmacia
Biotech) were run simultaneously with protein homogenates.
Ponceau red staining of blots was used to assess total protein
quality. Primary antibodies were rat reactive affinity-purified polyclonal IgG specific for TNF-␣ (1:250), IL-6 (1:200),
IGF-1 (1:100), TNF receptor (TNF-R)1 (1:150), IL-6 receptor
(IL-6-R)␣ (1:50), and IGF-1 receptor (IGF-1-R)␣ (1:100)
(Santa Cruz Biotechnology). Immunoblots were stained with
the appropriate secondary antibodies (Santa Cruz Biotechnology) and revealed with the enhanced chemiluminescence
system (Amersham Pharmacia Biotech). Primary antibody
for extracellular signal-regulated kinase (ERK)1 (1:500,
Santa Cruz Biotechnology) was used to correct for potential
differences in the amount of total protein loaded and transferred. Thus quantitative evaluation of specific protein expression was approached by scanning densitometry (imaging
densitometer model GT-670, Bio-Rad) of the exposed bands
normalized to the expression of ERK1 from the same lane.
Antibody positive controls were total proteins from endotoxin-stimulated (10 ␮g/ml for 8 h) rat macrophage cells (for
TNF-␣/TNF-R1 and IL-6/IL-6-R␣) and the normal rat liver
(IGF-1/IGF-1-R␣).
Immunohistochemical analysis. Immunohistochemistry
was performed on 4-␮m-thick sections from formalin-fixed
paraffin-embedded blocks of the left ventricle cross sectioned
perpendicularly to their major axis. Briefly, tissue immunoreactivity was intensified by microwave treatment in 10 mM
STRETCH-INDUCED MYOCARDIAL GROWTH FACTORS AND CYTOKINES EXPRESSION
AJP-Heart Circ Physiol • VOL
mained near identical up to 60 min. In comparison,
under severe stretch, a further and progressive increase in TNF-␣, IL-6, and IGF-1 levels was observed
up to 20 min, although, at that time, the enhancement
of gene expression was less evident for IL-6 (⬃1.2-fold
increase, P ⫽ NS) than for both TNF-␣ and IGF-1
(⬃3.1- and ⬃2.5-fold increase, respectively, both P ⬍
0.005). After 20 min of severe stretch, whereas TNF-␣
levels continued to increase, approaching a plateau
between 40 and 60 min (⬃4.7-fold increase vs. the
corresponding moderate stretch, P ⬍ 0.005), both IL-6
and IGF-1 levels progressively decreased. In particular, at 60 min of severe stretch, both IL-6 and IGF-1
levels were significantly lower than those observed
under moderate stretch (P ⬍ 0.005 and P ⬍ 0.05,
respectively), and IL-6 levels were even significantly
lower than those observed under control conditions
(P ⬍ 0.005).
Figure 2 shows representative autoradiograms (A)
and corresponding densitometry (B) of total myocardial
protein by Western blotting for TNF-␣, IL-6, and
IGF-1. In unstretched/unperfused hearts (data not
shown) and throughout the control perfusion (unstretched myocardium), TNF-␣ was undetectable,
whereas both IL-6 and IGF-1 were constitutively and
stably expressed. Moderate stretch resulted in de novo
TNF-␣ production, which was maximal after 10 min
and remained stable up to 60 min, whereas it had only
a marginal effect on IL-6 and IGF-1 expressions. When
the intraventricular balloon was inflated at the diastolic pressure of 35 mmHg, the effect of mechanical
stretch on myocardial TNF-␣, IL-6, and IGF-1 protein
production was more pronounced, and the patterns of
protein expression paralleled those of the corresponding mRNAs over time. Myocardial immunohistochemistry showed that the peptides were focally expressed
at the cardiomyocyte level (cytoplasmatic staining) and
mostly within the subendocardial wall layer (Fig. 3).
Figure 4 shows representative autoradiograms (A)
and corresponding densitometry (B) of total myocardial
protein by Western blotting for TNF-R1, IL-6-R␣, and
IGF-1-R␣. In unstretched/unperfused hearts (data not
shown) and throughout the control perfusion (unstretched myocardium), TNF-R1 and IGF-1-R␣ were
constitutively and stably expressed. No significant
changes in their expression occurred after myocardial
stretch regardless of the time and the level of the
balloon inflation, although TNF-R1 levels tended to be
progressively increased under severe stretch over time
(P ⫽ 0.072 vs. control at 60 min). IL-6-R␣ was not
detected in unstretched/unperfused hearts (data not
shown) or throughout the control perfusion (unstretched myocardium), nor it was expressed in the
myocardium from moderately and severely stretched
hearts until 60 min. Myocardial immunohistochemistry showed that TNF-R1 and IGF-1-R␣ were focally
expressed at the cardiomyocyte level (membrane staining) and diffusely within the myocardial wall (Fig. 5).
In parallel with Western blot analyses, TNF-R1 tended
to have a more intense immunostaining after 60 min of
severe stretch, which mainly localized within subendo-
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Fig. 1. A: representative Northern blots for tumor necrosis factor
(TNF)-␣, interleukin (IL)-6, insulin-like growth factor (IGF)-1, and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in control (unstretched myocardium: 5 mmHg), moderately (15 mmHg), and severely (35 mmHg) stretched myocardium (see METHODS for details).
Twenty-five micrograms of total RNA were loaded for each lane. The
first and second lane of each panel display signals obtained after 60
min in control and moderately stretched myocardium, respectively,
and they are representative of near-identical signals observed after
10, 20, and 40 min. Probe control lane refers to endotoxin-stimulated
(10 ␮g/ml for 8 h) rat macrophage cells (for TNF-␣ and IL-6) and the
normal rat liver (for IGF-1). B: densitometric analysis of autoradiographic bands, expressed as means ⫾ SE of 3 independent experiments for each value of time and stretch (ST). The degree of induction (fold induction) was referred to as an arbitrary number, defined
as 1, assigned to the level of expression estimated in control (C; for
IL-6 and IGF-1) or in 10-min moderately stretched (for TNF-␣)
myocardium. *P ⬍ 0.05 and **P ⬍ 0.005 vs. control; †P ⬍ 0.05 and
‡P ⬍ 0.005 vs. the corresponding 15-mmHg group.
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AJP-Heart Circ Physiol • VOL
cardial wall layer at cardiomyocyte levels. No specific
myocardial IL-6-R␣ immunoreactivity was found in
control nor it was detected in the stretched myocardium.
DISCUSSION
Major findings of the present study. The results of the
present study provide the first evidence of rapid, coordinate, and differential changes in myocardial TNF-␣,
IL-6, and IGF-1 mRNA and protein expression in response to moderate and severe acute mechanical stress
in the normal rat independent of ischemia and neurohormonal interference. The divergent pattern of myo-
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Fig. 2. A: representative Western blots for TNF-␣, IL-6, IGF-1,
and extracellular signal-regulated kinase (ERK)1 in control (unstretched myocardium: 5 mmHg), moderately (15 mmHg), and
severely (35 mmHg) stretched myocardium (see METHODS for details). Fifty micrograms of total protein were loaded for each lane.
The first and second lane of each panel display the signals obtained after 60 min in control and moderately stretch myocardium, respectively, and they are representative of near-identical
signals observed after 10, 20, and 40 min. Antibody control lane
refers to endotoxin-stimulated (10 ␮g/ml for 8 h) rat macrophage
cells (for TNF-␣ and IL-6) and the normal rat liver (for IGF-1). B:
densitometric analysis of autoradiographic bands, expressed as
means ⫾ SE of 3 independent experiments for each value of time
and stretch. The degree of induction was referred to as an arbitrary number, defined as 1, assigned to the level of expression
estimated in control (for IL-6 and IGF-1) or in 10-min moderately
stretched (for TNF-␣) myocardium. *P ⬍ 0.05 and **P ⬍ 0.005 vs.
control; †P ⬍ 0.05 and ‡P ⬍ 0.005 vs. the corresponding 15-mmHg
group.
Fig. 3. Representative photomicrographs showing myocardial TNF-␣,
IL-6, and IGF-1 immunoreactivity (see METHODS for details). A: focal
cytoplasmatic cardiomyocyte TNF-␣ protein immunostaining in 60-min
severely stretched myocardium (subendocardial wall layer). B: focal
cytoplasmatic cardiomyocyte IL-6 protein immunostaining in 20-min
severely stretched myocardium (subendocardial wall layer). C: focal
cytoplasmatic cardiomyocyte IGF-1 protein immunostaining in 20-min
severely stretched myocardium (subendocardial wall layer). Peroxidase/
diaminobenzidine (DAB) staining; original magnification of all photomicrographs, ⫻500.
STRETCH-INDUCED MYOCARDIAL GROWTH FACTORS AND CYTOKINES EXPRESSION
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courses would be consistent with the involvement
of specific transcriptional and/or posttranscriptional
mechanisms. To a certain extent, this specificity is also
supported by the immunohistochemical findings demonstrating that, in response to graded mechanical
stretch, myocardial TNF-␣, IL-6, and IGF-1 expression
were mainly modulated within the subendocardial wall
layer. This is consistent with the expected transmural
gradient of “normal” strains (fibers extension along the
circumferential, longitudinal, and radial axes), as described by Omens et al. (29) in an isolated, potassiumarrested dog heart model. Specifically, three-dimen-
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Fig. 4. A: representative Western blots for TNF receptor (TNF-R)1,
IL-6 receptor (IL-6-R)␣, IGF-1 receptor (IGF-1-R)␣ and ERK1 in
control (unstretched myocardium: 5 mmHg), moderately (15 mmHg),
and severely (35 mmHg) stretched myocardium (see METHODS for
details). Fifty micrograms of total protein were loaded for each lane.
The first and second lane of each panel display the signals obtained
after 60 min in control and moderately stretch myocardium, respectively, and they are representative of near-identical signals observed
after 10, 20, and 40 min. Antibody control lane refers to endotoxinstimulated (10 ␮g/ml for 8 h) rat macrophage cells (for TNF-R1 and
IL-6-R-␣) and the normal rat liver (for IGF-1-R␣). B: densitometric
analysis of autoradiographic bands (TNF-R1 and IGF-1-R␣), expressed as means ⫾ SE of 3 independent experiments for each value
of time and stretch. The degree of induction was referred to as an
arbitrary number, defined as 1, assigned to the level of expression
estimated in the control myocardium.
cardial TNF-␣, IL-6, and IGF-1 expression by graded
mechanical stretch, in the presence of substantially
unchanged GAPDH and ERK1 levels (used as internal
standards for Northern and Western blot analyses,
respectively), suggests that the differential gene expression occurred as a specific result of the different
magnitudes of mechanical stimulation. In particular,
the similarity between the mRNA and protein time
AJP-Heart Circ Physiol • VOL
Fig. 5. Representative photomicrographs showing myocardial TNFR1, IL-6-R␣, and IGF-1-R␣ immunoreactivity (see METHODS for details). A: focal, membrane, cardiomyocyte TNF-R1 protein immunostaining in 60-min severely stretched myocardium (subendocardial
wall layer). B: absence of IL-6-R␣ protein immunostaining in 60-min
severely stretched myocardium (subendocardial wall layer). Inset,
IL-6-R␣ protein immunostaining in rat transitional cell carcinoma,
used as a positive control (25). C: focal, membrane, cardiomyocyte
IGF-1-R␣ protein immunostaining in 60-min severely stretched myocardium (subendocardial wall layer). Peroxidase/DAB staining; original magnification of all photomicrographs, ⫻500.
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are, however, sufficient to determine important biological effects.
Possible mechanisms for changes in myocardial
TNF-␣, IL-6, and IGF-1 expression by graded mechanical stretch. It is known that once the mechanical
stimulus is received by specific mechanosensors (integrins, cytoskeleton, and sarcolemmal proteins), it is
converted into three major intracellular cross-talking
signal transduction pathways, i.e., the mitogen-activated protein kinase (MAPK), Janus kinase/STAT, and
calcineurin-dependent pathways, which ultimately
modulate gene expression through activation of disparate downstream nuclear transcription factors (14, 33).
Interestingly, it has been recently reported that the
activation of p38 (a member of the MAPK superfamily
belonging to the stress-activated protein kinases subfamily) leads to the activation of the transcription
factor nuclear factor-␬B (8), which is required for the
induction of most cytokine genes, including TNF-␣ and
IL-6 (2). The above signal transduction pathways may
be also activated in response to stretch-induced endogenous autocrine/paracrine cytokines and growth-promoting factors, which may act in a synergistic, antagonistic or permissive manner (14, 33). Given this
intricate scenario, it is possible that the differential
gene expression reported in the current study could
have occurred as a result of multiple levels of integration between the above cross-talking signal-transduction pathways.
Potential implications. Extrapolation of the present
acute in vitro observations to the chronic in vivo process of cardiac hypertrophy/remodeling and failure requires extreme caution. The two magnitudes of acute
myocardial stretch used in the current report (15 and
35 mmHg of end-diastolic pressure) were chosen as
representative left ventricular loads that may occur
after mild-to-moderate and severe myocardial infarction commonly associated with phenotypically and
prognostically different outcomes (28, 31, 32). In this
scenario, our results might be relevant for two reasons.
First, they suggest that cytokines and growth factors,
such as TNF-␣, IL-6, and IGF-1, may play an important role in the orchestration and timing of stretchinduced responses within the myocardium. In particular, the hypothesis could be put forward that the initial
response of the myocardium to moderately increased
hemodynamic load may be characterized by the contemporary activation, among others, of endogenous
autocrine/paracrine TNF-␣, IL-6, and IGF-1 aimed at
promoting functionally adaptive cardiac hypertrophy
to match the increased workload. Indeed, these peptides have shown to activate hypertrophic growth in
the cardiac myocyte (15, 18, 41) and to exert cytoprotective effects within the myocardium as well (8, 24, 26,
38). Conversely, under conditions of excessive hemodynamic stimulation, mechanical stress might subsequently promote and sustain an unbalanced milieu of
these peptides within the myocardium, with enhanced
generation of TNF-␣ accompanied by a simultaneous
reduction of IL-6 and IGF-1. This divergent expression,
by altering the local balance between growth and death
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sional myocardial normal strains, measured by biplane
radiography of transmural sets of radiopaque beads
implanted in the midanterior left ventricular free wall,
increase in proportion to left ventricular end-diastolic
pressure changes and from the subepicardium to subendocardium. Taken together, our observations suggest a differential relationship between mechanical
forces acting on cardiomyocytes and TNF-␣, IL-6, and
IGF-1 expression, thus complementing and extending
previous observations in similar ex vivo or in vitro
models of myocardial/cardiomyocyte stretch (9, 20, 30).
In agreement with previous studies, we found that
TNF-R1 and IGF-1-R, but not IL-6-R, were constitutively expressed by cardiomyocytes within the normal
(unstretched and/or unperfused) rat myocardium (4,
12, 17). Importantly, at variance with the corresponding peptides, the levels of myocardial TNF-R1, IL-6-R,
and IGF-1-R protein expression did not significantly
change in response to acute mechanical stretch challenges, indicating that the altered expression of the
peptides was not counterregulated at the receptor
level. However, it is interesting to note that the
myocardial TNF-R1 levels tended to be progressively
upregulated in response to severe stretch over time,
suggesting a possible enhancement of endogenous
autocrine/paracrine TNF system activity within the
stressed myocardium after persistent mechanical stimulation. Supporting this view are recent reports demonstrating the simultaneous increase in both myocardial TNF-␣ and TNF-R1 expression in end-stage
human heart failure (37) and after experimental myocardial infarction in the noninfarcted contralateral
wall (17). The absence of changes in myocardial IGF1-R protein expression in response to mechanical
stretch in our study contrasts with previous evidence
reporting a significant upregulation of IGF-1-R expression in overloaded left ventricular hypertrophy (9).
This discrepancy may be explained by the different
experimental model used and/or by the presence of
ischemic and/or systemic neurohormonal interference.
Finally, although no myocardial IL-6-R protein expression was detected up to 60 min of moderate and severe
stretch, we cannot exclude that IL-6-R is likely inducible and thus detectable after longer period of mechanical stimulation. Congruent with this hypothesis, a
recent study by Chandrasekar et al. (4) reported that
no postischemic (15 min) myocardial IL-6-R protein
expression was detected until 2 h of reperfusion. This
consideration notwithstanding, the biological significance of constitutive myocardial IL-6 expression, as
reported in our previous study (4), remains elusive. A
recent study by Craig et al. (8) showed that IL-6 confers significant protection against apoptosis in stressed
cultured cardiomyocytes. Although the authors demonstrated that this effect was associated with a robust
increase in downstream signal transducer and activator of transcription (STAT)3 activation, suggesting the
presence of constitutive IL-6R expression, no direct
demonstration was de facto provided. Taken together,
it may be speculated that myocardial IL-6-R is expressed at very low levels under basal condition, which
STRETCH-INDUCED MYOCARDIAL GROWTH FACTORS AND CYTOKINES EXPRESSION
We are grateful to Dr. Raffaela Pero, Dr. Francesca Lembo, and
Dr. Franco Fulciniti for expertise during the experimental staging of
this study and to Dr. Beatrice Ferravante for helpful discussions.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
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