Hypertrophy is one of the most important compensatory responses
of the myocardium, as it is critical for the maintenance of normal
cardiac function in response to pressure overload in the setting of
hypertension or volume overload during valvular insufficiency. However,
the mechanical stimuli of pressure versus volume overload
produce distinct cardiac muscle phenotypes, with the former resulting
in a concentric pattern of hypertrophy, and the latter promoting an
eccentric form of hypertrophy with cardiac chamber dilation. On a
single cell level, cardiac myocytes isolated from concentric
hypertrophic hearts display an increase in cell diameter with the
addition of new myofibrils in parallel(1, 2) , while
myocytes derived from dilated hearts exhibit an increase in cell
length, reflecting the addition of new sarcomeric units in
series(3, 4) . These distinct morphological phenotypes
are associated with different effects on global cardiac function.
Chronic volume overload hypertrophy can result in an irreversible loss
of cardiac function, while pressure overload hypertrophy is usually
associated with the preservation of contractile function, and can often
be reversible. While both forms of hypertrophy can lead to the
induction of the atrial natriuretic factor gene, distinct molecular
phenotypes have also recently been observed(5) . Although it
has long been accepted that there are distinct morphologic forms of
hypertrophy in the setting of pressure or volume overload, the
signaling pathways which mediate these distinct cardiac phenotypes
remain unclear. One of the critical questions is whether divergent
signaling pathways are responsible for the activation of these distinct
forms of hypertrophy that display clear differences with respect to
cell morphology, physiology, and the induction of molecular markers.
Our current insight into the mechanisms controlling cardiomyocyte
hypertrophy has primarily been obtained from an in vitro model
employing neonatal rat ventricular myocytes. A number of growth factors
signaling through G-protein coupled receptors, including
-adrenergic
agonists(6, 7, 8, 9, 10, 11) ,
endothelin 1(12, 13) , and angiotensin
II(14) , promote a hypertrophic response in this in vitro system. Following activation of the G-protein-dependent pathways,
cardiomyocytes display a uniform enlargement of cell size, with
increases in cell width and length, resulting from the assembly of new
myofibrils in parallel(12, 15, 16) . ras-dependent pathways appear to be both necessary and
sufficient to activate the hypertrophic response following
-adrenergic stimulation in the in vitro model
system(17) , and a recent study in transgenic mice documented
that the overexpression of a constitutively active ras protein
in the ventricular chambers results in a concentric form of cardiac
hypertrophy(18) . Taken together, these studies indicate that
G-protein coupled receptors and ras-dependent pathways
activate a form of cardiac hypertrophy that resembles the hypertrophy
seen in the setting of pressure overload. The question arises as to
whether divergent signaling pathways would mediate a volume overload
like hypertrophic phenotype, characterized by sarcomere assembly in
series and myocyte enlargement primarily due to an increase in cell
length versus width.
By coupling expression cloning to a
miniaturized version of the in vitro hypertrophy assay, we
have recently isolated a novel 21.5-kDa protein that induces an
increase in cell size and atrial natriuretic factor (ANF) (
)release in cardiomyocytes(19) . The protein was
designated cardiotrophin-1 (CT-1). Sequence similarity data and
structural considerations suggest that CT-1 is a new member of a family
of structurally related cytokines including interleukin (IL)-6 and
IL-11, leukemia-inhibitory factor (LIF), ciliary neurotrophic factor
(CNTF), and oncostatin M (OSM). The receptors of this cytokine family
are multimeric and share the class-specific transmembrane signal
transducing component gp130 (20, 21, 22, 23) . Signaling is
triggered through the homodimerization of gp130 (24) , or the
heterodimerization of gp130 with a related transmembrane signal
transducer, the LIF receptor subunit
(LIFR
(25, 26) . The gp130/LIFR
heterodimer
constitutes the functional bipartite LIF receptor
complex(22, 27) . Other members of this cytokine
family first bind to a private receptor component and subsequently
induce gp130 dimerization. For example, IL-6 binds to the receptor
component IL-6R, and the IL-6
IL-6R complex then induces the
homodimerization of gp130 (28, 29) ; CNTF binds to the
receptor component CNTFR
and induces gp130/LIFR
heterodimerization(26, 30, 31) .
Consequently, the expression pattern of the various receptor components
defines the spectrum of action of different members of the gp130
cytokine family.
The cloning of CT-1 based on its ability to induce
an increase in cell size in cardiomyocyte culture (19) suggested that gp130-dependent signaling pathways may be
coupled to cardiomyocyte hypertrophy. In the present study we define
the receptor system used by CT-1 in cardiomyocytes, and we examine the
effects of CT-1 and two other members of the IL-6 cytokine family, LIF
and CNTF, on morphological and molecular features defining a
hypertrophic response in cultured cardiomyocytes. By using a monoclonal
anti-gp130 antibody and a LIFR
antagonist, we establish that both
gp130 and LIFR
are required for CT-1 signaling in cardiomyocytes.
Immunoprecipitation and immunoblotting data show that CT-1 induces the
tyrosine phosphorylation of gp130 and a higher molecular weight
protein, corresponding in size to LIFR
. Using confocal laser
microscopy of cells stained for both thick and thin filament markers,
our studies further indicate that gp130/LIFR
-dependent signaling
pathways induce a hypertrophic phenotype in cardiomyocytes that is
distinct from the response seen after
-adrenergic stimulation, and
that shows resemblance to volume overload cardiac hypertrophy both with
regard to cell morphology and gene expression pattern.
EXPERIMENTAL PROCEDURES
Murine LIF was produced at Genentech, Inc. Murine LIF was
used throughout the study, except for the experiment employing the
LIFR
antagonist (hLIF-04), in which both murine LIF and human LIF
were used. Rat CNTF and human IL-6 were purchased from Boehringer
Mannheim and from Genzyme, respectively. Human sIL-6R was prepared as
outlined previously(21) . The
-adrenergic agonist
phenylephrine (PE) was obtained from Sigma.
Expression and Purification of the CT-1 Fusion
Protein
The reading frame of murine CT-1 was cloned C-terminal
to the sequence encoding the herpes simplex virus glycoprotein D
followed by a factor Xa cleavage site. Following expression in 293
cells and cleavage of the herpes simplex virus secretion signal
sequence, the CT-1 fusion protein contained a 34-amino acid N-terminal
extension followed by the CT-1 sequence(19) . The CT-1 fusion
protein was purified from conditioned medium with a herpes simplex
virus glycoprotein D-specific monoclonal antibody and quantified by a
colorimetric assay (Bio-Rad).
LIFR
Antagonist hLIF-04
The LIFR
antagonist hLIF-04 was used to assess the requirement of the LIFR
for CT-1 signaling. Human LIF (hLIF) and the hLIF mutant, hLIF-04, were
expressed as glutathione S-transferase fusion proteins, as
described previously(32) . hLIF-04 contains three amino acid
substitutions (Q25A, S28A, and Q32A), within the A-helix of the hLIF
molecule, resulting in a selective disruption of the gp130 binding
site, but no impairment in LIFR
binding. A detailed
characterization of the LIFR
antagonist hLIF-04 will be presented
elsewhere. (
)
Cell Culture
Hearts from
1-3-day-old Sprague-Dawley rats were removed, the ventricles were
pooled, and the ventricular cells were dispersed by digestion with
collagenase II (Worthington) and pancreatin (Life Technologies, Inc.).
The cell suspension was purified by centrifugation through a
discontinuous Percoll gradient to obtain myocardial cell cultures with
>95% myocytes, as assessed by immunofluorescence with an
anti-TrpE/MLC-2v antiserum(12, 33) . The
cardiomyocytes were plated at a density of 5-6
10
cells/cm
in Dulbecco's modified Eagle's
medium (DMEM)/medium 199 (4:1) supplemented with 10% horse serum, 5%
fetal bovine serum, glutamine and antibiotics (plating medium), and
allowed to attach overnight. The cells were then washed twice with
DMEM/medium 199 and further incubated in DMEM/medium 199 supplemented
with glutamine and antibiotics (maintenance medium) in the presence or
absence of various agents. Neonatal mouse ventricular myocytes
were isolated from 1-4-day-old NIH Swiss mice, following the
protocol outlined above. One litter (8-12 pups) yielded
5
10
cells. Approximately 95% of the cells displayed
spontaneous contractile activity in culture.
Immunoprecipitation and
Immunoblotting
Cardiomyocytes were stimulated with various
agents and solubilized with ice cold lysis buffer (0.5% Nonidet P-40,
150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 5 mM EDTA, 2 mM sodium vanadate, 1 mMp-amidinophenylmethanesulfonyl fluoride (Sigma), 5
µg/ml aprotinin). Clear lysates were obtained by centrifugation and
immunoprecipitated with a polyclonal rabbit anti-gp130 cytoplasmic
peptide antibody(34) . Immunoprecipitates (protein A-Sepharose,
Pharmacia Biotech Inc.) were subjected to SDS-PAGE under reducing
conditions and immunoblotted with an anti-phosphotyrosine antibody
(4G10, Upstate Biotechnology), employing an enhanced chemiluminescence
detection system (Amersham Corp.). For the detection of protein
tyrosine phosphorylation, the cell lysates were analyzed directly by
SDS-PAGE and immunoblotting with 4G10.
Immunofluorescence Techniques and Morphometric
Analysis
Cardiomyocytes were plated in Lab-Tek plastic chamber
slides (Nunc), precoated with 4 µg/cm
laminin (Sigma),
and incubated in the presence or absence of various agents. After 48 h,
the cells were rinsed with phosphate-buffered saline, fixed in 4%
paraformaldehyde, and permeabilized with 0.3% Triton X-100. Following
three phosphate-buffered saline washes, the chamber slides were
incubated in 3% bovine serum albumin for 10 min to block nonspecific
sites. Subsequently, the cells were dual-stained for 1 h in 3% bovine
serum albumin with 1) a polyclonal anti-TrpE/MLC-2v antiserum from
rabbit(12) , combined with a monoclonal mouse anti-rat ANF
antibody (35, 36) , or 2) a monoclonal mouse
anti-human
myosin heavy chain (
MHC) antibody(37) ,
combined with rhodamine phalloidine (Molecular Probes). Following four
phosphate-buffered saline washes, the slides were blocked with 5%
normal donkey serum for 10 min, incubated for 1 h in 5% normal donkey
serum using a Texas Red-conjugated donkey anti-rabbit IgG and a
fluorescein isothiocyanate-conjugated donkey anti-mouse IgG as
secondary antibodies (Jackson Laboratories), and finally mounted on
glass coverslips. Cardiomyocytes stained against MLC-2v and ANF were
viewed by fluorescence microscopy. Cell size was estimated by measuring
the area to which individual MLC-2v-positive cells attached
(planimetry), cell length and width were determined as outlined in Fig. 1, and the percentage of MLC-2v positive cells that
displayed perinuclear staining for immunoreactive ANF was calculated.
Cardiomyocytes stained against
MHC and F-actin (rhodamine
phalloidine) were analyzed by confocal laser microscopy using a 60
oil immersion objective. Images of cardiomyocytes stained
against
MHC were used to determine the effect of various agonists
on the average sarcomere length.
Figure 1:
Definition
of morphometric parameters. Cell length was defined as the maximum
longitudinal extension of individual cells. Maximum cell width was
measured perpendicular to the axis defining cell
length.
ANF Radioimmunoassay
The concentration of
immunoreactive ANF in cell culture supernatants was determined by
radioimmunoassay, using the monoclonal mouse anti-rat ANF antibody with
synthetic ANF-(99-126) as the standard and
I-ANF-(99-126)
(Amersham) as the trace(35) .
Isolation and Hybridization of RNA
Total cellular
RNA was isolated by a modification of the acid guanidium/thiocyanate
phenol/chloroform extraction method (38) using the RNA STAT 60
RNA isolation reagent (Tel-Test ``B''). RNA was
size-fractionated by formaldehyde agarose gel electrophoresis,
transferred to nylon filters (Magna NT) by overnight capillary
blotting, and hybridized with cDNA probes labeled with
[
-
P]dCTP by random priming (Life
Technologies, Inc.). The filters were washed under stringent conditions
and exposed to x-ray film (Kodak X-Omat AR). Signal intensities were
determined by densitometry (UltroScan XL, Pharmacia). The following
cDNA probes were used: mouse c-fos (1.2-kb
fragment)(39) , mouse egr-1 (zif/268) (1.7-kb
fragment(40) , human c-myc (1.6-kb fragment, a
generous gift of Dr. J. Feramisco), mouse c-jun (2.6-kb
fragment)(41) , mouse jun-B (1.90-kb
fragment)(42) , mouse tis11 (1.0-kb fragment, a
generous gift of Dr. H. R. Herschman)(43) , rat prepro-ANF (0.6
kb of coding region)(44) , rat skeletal
-actin (0.2 kb of
a 3`-untranslated region generated by reverse transcriptase-polymerase
chain reaction from skeletal muscle poly(A)
RNA) (45) and rat MLC-2v (0.6 kb of coding region) (46) .
Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1.2-kb
fragment) (47) and mouse 28s (5-kb fragment) cDNA probes were
used to control for loading and transfer efficiency.
Nuclear Run-on Transcription Assay
Run-on
transcription assays were performed as described
previously(48) . Cardiomyocytes were lysed in ice-cold Nonidet
P-40 buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3
mM MgCl
, 0.5% Nonidet P-40) and further disrupted
by 10 gentle strokes with a loose pestle in a Dounce homogenizer. The
nuclei were recovered by centrifugation. The supernatants (representing
the cytosolic fractions) were saved, and vanadylribonucleoside
complexes (Life Technologies, Inc.) were added to inhibit RNase
activity. Total RNA was isolated from the cytosolic fractions and
subjected to Northern blot analysis using prepro-ANF, GAPDH, and 28s
cDNA probes. The nuclei were washed once in Nonidet P-40 buffer,
resuspended in 100 µl of storage solution (50% glycerol, 50 mM HEPES, pH 7.4, 5 mM MgCl
, 0.1 mM EDTA, 5 mM dithiothreitol), and stored at -80
°C. On the day of the assay, the nuclei were thawed, 27.5 µl of
run-on buffer (25 mM Tris-HCl, pH 7.5, 12.5 mM MgCl
, 750 mM KCl, 25 mM dithiothreitol, 1.25 mM of ATP, GTP, and CTP), 6 µl
(62.5 µCi) of [
-
P]UTP, and 1 µl of
RNasin (Promega) were added, and the nuclei were incubated at 30 °C
for 30 min. Following DNA and protein digestion (RQ-1 DNase 1, Promega;
proteinase K, Life Technologies, Inc.), the
P-labeled RNA
was extracted once with phenol/chloroform, ethanol-precipitated, and
purified over a RNase-free Sephadex G50 column (Boehringer Mannheim).
The total amount of
P-labeled RNA recovered from each
reaction was determined by liquid scintillation counting (Beckman).
Five micrograms of linearized, alkali-denatured pBluescript
(Stratagene), pBluescript harboring the rat prepro-ANF
cDNA(44) , and pBluescript harboring the human GAPDH cDNA (47) were spotted onto nylon filters in advance. The filters
were prehybridized for 1 h in 56.25% formamide, 0.25 M sodium
phosphate, pH 7.2, 0.25 M NaCl, 1 mM EDTA, 7% SDS. An
equal number of counts from each run-on reaction were added to the
prehybridization solution, and hybridizations were carried out for 48 h
at 42 °C. Finally, the filters were washed under stringent
conditions and exposed to x-ray film. The amount of ANF and
GAPDH-specific hybridization was determined by densitometry. No
hybridization to the empty pBluescript control was detectable (data not
shown).
Plasmid Constructs
pANF(-3003)Luc5`, an
ANF-luciferase reporter construct composed of the most proximal 3003 bp
of the rat ANF gene 5`-flanking region inserted into the promoterless
firefly luciferase reporter plasmid pSVOALuc5` was used to assess the
CT-1-, LIF-, CNTF-, and PE-induced activity of the rat ANF
promoter(11) . pRSVLuc5`, a RSV-luciferase reporter construct,
and pSVOALuc5` served as positive and negative controls,
respectively(49) . To correct for transfection efficiency,
cardiomyocytes were cotransfected with pON249, a
-galactosidase
expression vector under the control of the human cytomegalovirus
promoter(50) .
Transfection and Luciferase/
-Galactosidase
Assays
Cells were plated in 3.5-cm dishes in duplicate, allowed
to attach overnight, and switched to maintenance medium supplemented
with 4% horse serum. Two hours later, the cells were cotransfected with
2.75 µg of either pANF(-3003)Luc5`, pRSVLuc5`, or pSVOALuc5`,
and 0.75 µg of pON249 using the calcium phosphate method as
described by Chen and Okayama (51) . After 18-20 h, the
cells were washed twice to remove the fine layer of calcium precipitate
and incubated in maintenance medium in the presence or absence of
various agents. At different time points, the cells were washed twice
with ice-cold phosphate-buffered saline and were lysed for 30 min on
ice in 100 µl of lysis buffer (0.1 M KH
PO
, pH 7.9, 0.5% Triton X-100, 1 mM dithiothreitol). Luciferase activities were determined in
duplicate samples from each plate; 20 µl of cell lysate were
combined with 100 µl of luciferase assay buffer (100 mM Tricine, pH 7.8, 10 mM MgSO
, 2 mM EDTA, 2 mM ATP, 1 mM dithiothreitol, 73
µM
-luciferin), and luciferase activities were
measured using a Monolight 401 luminometer (Analytical Luminescence
Laboratory).
-Galactosidase activities were determined in
duplicate samples from each plate; 20 µl of cell lysate were added
to 250 µl of
-galactosidase assay buffer (0.1 M sodium phosphate, pH 7.3, 1.2 mM MgCl
, 60
mM
-mercaptoethanol, 1.8 mM chlorophenol
red-
-D-galactopyranoside (Boehringer Mannheim)), and
incubated at 37 °C for 15 min.
-Galactosidase activities were
obtained by measuring OD at 574 nm, using lysis buffer as a blank.
Statistical Analysis
Data are presented as means
± S.E. p values were determined using one-way analysis
of variance.
RESULTS
Induction of a Distinct Pattern of Immediate Early
Genes following CT-1 versus
-Adrenergic Stimulation of Cultured
Myocardial Cells
The induction of immediate early (I.E.) genes
precedes the late phenotypic changes in cardiomyocytes following the
stimulation of G-protein-coupled receptors in vitro(12, 13, 14, 15) and cardiac
hypertrophy in vivo(52, 53) . We therefore
determined whether stimulation of cardiomyocytes with CT-1, LIF, or
CNTF would induce I.E. gene expression (Fig. 2). Stimulation
with CT-1 resulted in the rapid and transient induction of a panel of
I.E. genes, including c-fos, egr-1, c-myc,
c-jun, jun-B, and tis11. The pattern of I.E.
gene expression in response to stimulation with LIF was virtually
indistinguishable from the pattern induced by CT-1. CNTF displayed a
relatively weak induction of the same panel of I.E. genes as did CT-1
and LIF. As compared to stimulation with PE, CT-1, and LIF induced
>10 fold higher levels of c-myc and tis11.
Figure 2:
Immediate early gene induction. Neonatal
rat ventricular cardiomyocytes were plated into 15-cm dishes and
serum-starved for 18 h. Thereafter, control cells were harvested; the
remaining cells were stimulated with PE (100 µM), CT-1,
LIF, or CNTF (1 nM each), for the indicated times. Total RNA
was isolated and subjected to Northern blot analysis (10 µg/lane).
The following probes were used: c-fos, egr-1,
c-myc, c-jun, jun-B, tis11, and
28s. Data from one experiment are presented. One additional experiment
yielded comparable results.
Involvement of gp130 and LIFR
in CT-1 Induction of
c-fos
Previous studies have employed blocking antibodies
directed against gp130 to demonstrate that IL-6, LIF, OSM, CNTF, and
IL-11 utilize the common receptor component
gp130(21, 22, 23) . Sequence similarity data,
as well as structural considerations, suggest that CT-1 might be a new
member of this cytokine family, and might therefore share the receptor
component gp130(19) . To directly test this hypothesis, we
examined the effect of an anti-gp130 antibody on the CT-1 induction of
c-fos in cardiomyocytes. A monoclonal anti-gp130 antibody
(RX435) was generated by immunizing rats with recombinant murine gp130. (
)RX435 specifically blocks the action of LIF, IL-6, IL-11,
and OSM on mouse myeloid leukemic M1 cells
and inhibits the
binding of CT-1 to M1 cells(54) . Since RX435 does not
cross-react with rat gp130, we used neonatal mouse cardiomyocytes in
this set of experiments. Similar to the rat system, murine
cardiomyocytes responded to stimulation with CT-1, LIF, and PE with an
induction of immediate early genes and typical morphology changes.
However, CT-1, LIF, and PE did not induce ANF gene expression in murine
cardiomyocytes (data not shown). This might relate to the fact that in
mice, in sharp contrast to rats, the ventricular expression of ANF is
not down-regulated after birth, but instead remains at high levels from
embryogenesis through adulthood (55) . We first examined the
effects of different concentrations of CT-1, LIF and PE on the
induction of c-fos in murine cardiomyocytes. CT-1, LIF, and PE
induced c-fos expression in a dose-dependent manner (Fig. 3, A and B). In subsequent experiments,
cardiomyocytes were incubated for 1 h with RX435 or, as a control, with
immunopurified rat IgG prior to stimulation with CT-1, LIF, or PE.
RX435 was used at a concentration that has previously been shown to
inhibit
80% of CT-1 (0.12 nM) binding to murine M1
cells(54) . CT-1, LIF, and PE were used at concentrations that
resulted in an approximately 4-5-fold induction of c-fos expression. As shown in Fig. 3, C and D,
RX435 completely abolished the induction of c-fos by CT-1 and
LIF. By contrast, the c-fos induction by PE was not abolished
by RX435 (although somewhat suppressed), confirming the specificity of
the antibody (Fig. 3D).
Figure 3:
Effects of an anti-gp130 antibody and a
LIFR
antagonist on the c-fos induction by CT-1, LIF, and
phenylephrine. Neonatal mouse ventricular cardiomyocytes were plated
into 3.5-cm dishes and serum-starved for 18 h. Thereafter, the cells
were stimulated for 45 min with various concentrations of CT-1, LIF, or
PE (Panels A and B). In a second set of experiments,
cells were incubated with a monoclonal rat anti-mouse gp130 antibody
(RX435, 20 µg/ml) or immunopurified rat IgG (20 µg/ml) prior to
CT-1 (0.1 nM) stimulation (Panel C), and LIF (0.1
nM) or PE (100 µM) stimulation (Panel
D). In a third experiment (Panel E), cells were incubated
with a LIFR
antagonist (hLIF-04, 0.5 nM), prior to
stimulation with PE (100 µM), human LIF (hLIF),
murine LIF (mLIF), and CT-1 (0.1 nM each). Total RNA
was isolated and subjected to Northern blot analysis (2 µg/lane).
Equal loading and transfer conditions were confirmed by GAPDH and 28s
(not shown) hybridization.
To determine whether
LIFR
is required for CT-1 signaling, we studied the effect of a
LIFR
antagonist, hLIF-04, on c-fos induction by CT-1.
hLIF-04 was generated by introducing three amino acid substitutions in
the human LIF molecule, thereby abrogating the gp130 binding site.
hLIF-04 has been shown to antagonize the action of hLIF in a Ba/F3 cell
line, stably transfected with human gp130 and LIFR
(see
``Experimental Procedures''). As shown in Fig. 3E, preincubation with hLIF-04 blocked the
c-fos induction by human LIF, murine LIF, and by CT-1. By
contrast, c-fos induction by PE was not affected by hLIF-04.
Taken together, the results obtained with the anti-gp130 blocking
antibody and the LIFR
antagonist strongly suggest that CT-1, like
LIF, requires both gp130 and LIFR
to activate downstream signaling
pathways leading to the induction of c-fos in cardiomyocytes.
CT-1 and LIF Induce Tyrosine Phosphorylation of gp130 and
a 200-kDa Protein in Cardiomyocytes
To examine whether CT-1
would induce tyrosine phosphorylation of gp130, as it has been shown
for the other members of the IL-6 cytokine family, cardiomyocytes were
stimulated with soluble IL-6R, IL-6, a combination of IL-6 and sIL-6R,
LIF, CT-1, and PE. Cell lysates were immunoprecipitated with a
polyclonal anti-gp130 antiserum, analyzed by SDS-PAGE, and
immunoblotted with an anti-phosphotyrosine antibody. As shown in Fig. 4A, the IL-6/sIL-6R complex, LIF, and CT-1 induced
gp130 tyrosine phosphorylation. By contrast, no gp130 phosphorylation
was detectable after stimulation with PE, and with IL-6 or sIL-6R
alone. To compare the pattern of protein tyrosine phosphorylation
induced by the various agonists, cell lysates were subjected to
SDS-PAGE and immunoblotting with the anti-phosphotyrosine antibody (Fig. 4B). Stimulation with the IL-6/sIL-6R complex,
LIF, and CT-1 resulted in the tyrosine phosphorylation of a protein,
corresponding in size to gp130 (compare to Fig. 4A). An
additional, higher molecular mass protein (approximately 200 kDa) was
tyrosine-phosphorylated in response to LIF and CT-1 only, but not in
response to the IL-6
sIL-6R complex.
Figure 4:
Protein tyrosine phosphorylation. Neonatal
rat ventricular cardiomyocytes were plated into 15-cm dishes and
serum-starved for 18 h. Thereafter the cells were stimulated with
sIL-6R, IL-6, IL-6 and sIL-6R combined, LIF, and CT-1 (10 nM each), or PE (100 µM) for 10 min at 37 °C. Cell
lysates were immunoprecipitated with an anti-gp130 antibody, subjected
to SDS-PAGE, and immunoblotting with an anti-phosphotyrosine antibody
as outlined under ``Experimental Procedures'' (Panel
A). In addition, cell lysates were directly subjected to SDS-PAGE
and immunoblotting with an anti-phosphotyrosine antibody (Panel
B).
CT-1 Induces a Morphologically Distinct Hypertrophic
Response Characterized by the Assembly of Sarcomeric Units in
Series
Neonatal rat ventricular cardiomyocytes respond to
various hypertrophic stimuli by an increase in individual cell size,
assembly of myofibrils, and perinuclear accumulation of ANF. To assess
the effects of gp130/LIFR
-dependent cytokines on these
morphological features of a hypertrophic response, cardiomyocytes were
treated with CT-1, LIF, or CNTF and dual-stained with an
anti-TrpE/MLC-2v antiserum and a monoclonal anti-ANF antibody.
Unstimulated cells and PE-treated cells served as negative and positive
controls, respectively. The effects of CT-1, LIF, CNTF, and PE on
cardiomyocyte area, length, and width and the percentage of cells
displaying perinuclear accumulation of ANF are summarized in Table 1A. CT-1 and LIF induced a hypertrophic response, as
evidenced by increases in cardiomyocyte area and the percentage of
cells with perinuclear accumulation of immunoreactive ANF. As compared
to PE stimulation, CT-1 and LIF had a more pronounced effect on
cardiomyocyte length but did not significantly affect cardiomyocyte
width. Treatment of cardiomyocytes with CNTF did not induce significant
morphology changes as compared to control. Cardiomyocytes were
dual-stained with a monoclonal anti-
MHC antibody and rhodamine
phalloidine, to allow a simultaneous assessment of thick filament
(
MHC) and thin filament (F-actin) assembly(56) . Images
were obtained by confocal laser microscopy. Representative high power
fields are depicted in Fig. 5. Cardiomyocytes stimulated with
CT-1 (Panels E and F) and LIF (Panels G and H) displayed a high degree of sarcomeric organization:
myofibrils were oriented along the longitudinal cell axis, and extended
into the tips of the cytoplasmic projections. As compared with PE
stimulation (Panels C and D), sarcomeric units were
assembled predominantly in series, rather than in parallel (most
striking in cytoplasmic projections containing only a few myofibrils
(see Panels E-H)). As shown in Table 1B, the
average sarcomeric length did not differ significantly among the
experimental groups.
Figure 5:
Sarcomeric organization. Neonatal rat
ventricular cardiomyocytes were plated into chamber slides and
incubated for 48 h with no additions (Panels A and B), 100 µM PE (Panels C and D),
1 nM CT-1 (Panels E and F), 1 nM LIF (Panels G and H), or 1 nM CNTF (Panels I and J). Cells were dual-labeled with an
anti-
MHC antibody (Panels A, C, E, G, and I) and rhodamine phalloidine (Panels
B, D, F, H, and J), and viewed
by confocal laser microscopy. The bar (Panel A)
represents 10 µm.
CT-1 Induces ANF Gene Expression
The reactivation
of an embryonic pattern of gene expression is a hallmark of
cardiomyocyte hypertrophy. We therefore determined whether stimulation
of cardiomyocytes with CT-1, LIF, or CNTF would induce embryonic gene
expression (Fig. 6). As shown previously(11) ,
stimulation of neonatal rat ventricular cardiomyocytes with PE resulted
in a significant (5.1-fold) increase in prepro-ANF mRNA levels.
Likewise, treatment with CT-1 or LIF stimulated prepro-ANF mRNA
expression (5.1- and 5.0-fold, respectively). By contrast, CNTF did not
significantly affect prepro-ANF mRNA levels. As previously reported (8) , PE stimulation resulted in an increase (4.5-fold) in
skeletal
-actin mRNA expression. CT-1, LIF, and CNTF, however, did
not significantly affect skeletal
-actin steady state mRNA levels.
Finally, we examined the effects of CT-1, LIF, CNTF, and PE on the
expression of MLC-2v, a constitutively expressed contractile protein
gene. PE stimulation resulted in a marginal 1.5-fold induction of
MLC-2v mRNA levels. By contrast, CT-1, LIF, and CNTF did not induce
MLC-2v mRNA expression (data not shown). We next determined whether the
increase in prepro-ANF mRNA expression in response to CT-1 and LIF is
accompanied by an increase in secretion of immunoreactive ANF. As noted
in the initial study reporting the cloning of CT-1(19) , CT-1
induced ANF release from cardiomyocytes in a dose-dependent manner (Fig. 7). Likewise, LIF increased the secretion of ANF; maximum
ANF release was observed with CT-1 and LIF concentrations of
0.1-10 nM. Consistent with the ANF Northern blot
results, stimulation with CNTF did not induce ANF release at any of the
concentrations tested (Fig. 7).
Figure 6:
Expression of prepro-ANF and skeletal
-actin mRNAs. Neonatal rat ventricular cardiomyocytes were plated
into 15-cm dishes and incubated for 48 h with no additions (control), PE (100 µM), CT-1, LIF, or CNTF (1
nM each). Total RNA was isolated and subjected to Northern
blot analysis (10 µg/lane) using prepro-ANF and skeletal
-actin cDNA probes. Equal loading and transfer conditions were
confirmed by GAPDH and 28s hybridization. Representative blots are
presented in Panel A. The results from three independent
experiments, expressed as -fold induction over control, are depicted in Panels B and C. Values are means ± S.E.;
*p < 0.05,**p < 0.01 versus control.
Figure 7:
Release of immunoreactive ANF from
myocardial cells. Neonatal rat ventricular cardiomyocytes were cultured
in 24- well plates, and stimulated for 48 h with various concentrations
of CT-1, LIF, and CNTF. ANF concentrations in the culture supernatants
were determined by radioimmunoassay. Values are means ± S.E.
from triplicate wells. Supernatants from unstimulated control cells and
PE (100 µM) stimulated cells contained 2.5 ± 0.4
nM and 20.8 ± 1.5 nM ANF, respectively. Two
additional experiments yielded comparable
results.
CT-1 Activates the Transcription of the ANF
Gene
To determine whether the induction of ANF by CT-1 results
from an increased ANF gene transcription, we performed nuclear run-on
transcription assays. We compared the ANF gene transcription rate
during stimulation with CT-1 and PE. A typical experiment is presented
in Table 2and Fig. 8. Cardiomyocyte nuclei were isolated
at various time points during stimulation with CT-1 or PE. Unstimulated
cells served as a control. CT-1 and PE increased the overall RNA
transcription rate, as measured by the total amount of
P
incorporation into purified run-on RNA (Table 2). The overall RNA
transcription rate was higher at the early time points (0, 6, and 12
h), i.e. shortly after serum withdrawal (Table 2). An
equal number of cpm from each run-on reaction was hybridized to
denatured prepro-ANF and GAPDH cDNAs immobilized onto nylon filters.
The ANF autoradiograms are shown in Fig. 8A. The CT-1
and PE induced ANF transcription rates were calculated as -fold
induction over the corresponding control values from each time point (Fig. 8B). ANF transcription reached a maximum 2.0-fold
(CT-1) and 2.1-fold (PE) increase versus control at 24 h and
returned to control levels at 48 h. By contrast, GAPDH gene
transcription was not affected by CT-1 or PE (not shown). The CT-1- and
PE-induced increase in ANF gene transcription was accompanied by a
time-dependent accumulation of prepro-ANF mRNA in the cytosol, reaching
maximum levels between 24 and 48 h (Fig. 8C). By
contrast, CT-1 and PE did not affect GAPDH mRNA expression (not shown).
Figure 8:
CT-1- and phenylephrine-induced ANF gene
transcription. Same experiment as described in Table 2. 1.2
10
cpm from each run-on reaction were hybridized to
linearized and denatured pBluescript harboring the prepro-ANF cDNA,
immobolized onto nylon filters. The filters were washed under stringent
conditions, and exposed to x-ray film for 5 days (Panel A).
The signal intensities were analyzed by densitometry to quantitate the
amount of ANF gene transcription; data are presented as -fold induction
over control (Panel B). Total RNA was isolated from the
cytosolic fractions of the same cells, that were used for the run-on
assays. RNA was subjected to Northern blot analysis (7.5 µg/lane),
using prepro-ANF and 28s cDNA probes (Panel C). Data from one
experiment are presented. One additional experiment yielded comparable
results.
CT-1 Responsive Cis-regulatory Sequences Are Located
Outside a 3003-bp ANF Promoter Fragment
The transcriptional
up-regulation of the ANF gene in response to a number of G-protein
coupled receptor agonists, including PE, is mediated by cis-regulatory elements, which are located within the proximal
3 kb of ANF 5`-flanking region(11, 12, 57) .
We performed transient transfection analyses to determine whether
gp130/LIFR
-dependent cytokines and PE utilize common cis-acting elements within the ANF promoter region.
Cardiomyocytes were transfected with an ANF-luciferase reporter
construct containing 3003 bp of the rat ANF 5`-flanking region.
Cardiomyocytes transfected with a RSV-luciferase construct or a
promoterless luciferase construct were used as positive and negative
controls, respectively. To control for transfection efficiency, cells
were cotransfected with a
-galactosidase expression vector. The
cells were then stimulated with PE, CT-1, LIF, or CNTF; unstimulated
cells served as controls. At various time points, cardiomyocytes were
harvested for luciferase and
-galactosidase assays. As previously
reported(11) , the 3003-bp ANF promoter fragment conferred
PE-inducible expression to the luciferase reporter gene (maximum
23.7-fold induction at 48 h) (Fig. 9). By contrast, the 3003-bp
ANF promoter fragment did not mediate inducible expression by CT-1,
LIF, or CNTF (Fig. 9). Luciferase activities in cells
transfected with the RSV-luciferase construct were on average 80-fold
higher as compared to unstimulated cells transfected with the
ANF-luciferase construct and were not induced by any of the treatments
(not shown). Cells transfected with the promoterless reporter construct
displayed luciferase activities that were not different from background
values and were not affected by any of the treatments (not shown).
Considering that CT-1 as well as PE increase the ANF gene transcription
rate (as demonstrated by nuclear run-on assays), the transfection
analyses suggest that, in contrast to PE, the CT-1 responsive cis-regulatory elements are located outside of 3 kb of ANF
5`-flanking region. Thus, divergent pathways appear to mediate the
induction of the ANF gene in response to CT-1 as compared to
-adrenergic stimulation.
Figure 9:
Transient transfection analysis of a 3003-
bp ANF promoter-luciferase reporter construct. Neonatal rat ventricular
cardiomyocytes were cotransfected with an ANF-luciferase reporter
construct (pANF(-3003)Luc5`) and a
-galactosidase control
vector (pON249), and incubated with no additions (control), PE
(100 µM), CT-1, LIF, or CNTF (1 nM each). At the
indicated time points, cells were harvested for luciferase and
-galactosidase assays. Luciferase activities were normalized to
-galactosidase activities. Results are presented as -fold
induction over control cells harvested at corresponding time points.
Values are means ± S.E. of four experiments at each time point;
*p < 0.01 versus control.
DISCUSSION
CT-1 Signaling through the gp130/LIFR
Heterodimer
Cardiotrophin-1 was recently isolated by expression
cloning based on its ability to induce an increase in cell size in
cardiomyocyte culture(19) . The deduced amino acid sequence
suggested that CT-1 is a novel member of the IL-6, IL-11, LIF, CNTF,
and OSM family of structurally related cytokines, that trigger
downstream signaling pathways in multiple cell types through the
homodimerization of gp130 or the heterodimerization of gp130 and
LIFR
(22, 24, 26, 27) . To
determine whether CT-1 shares the receptor components gp130 and/or
LIFR
with the other members of the IL-6 cytokine family, we
employed a monoclonal anti-gp130 antibody and a mutated human LIF
protein, acting as a LIFR
antagonist. The c-fos induction
by CT-1 and LIF in cardiomyocytes was antagonized by the monoclonal
anti-gp130 antibody as well as the LIFR
antagonist, strongly
suggesting that CT-1, like LIF, utilizes the receptor components gp130
and LIFR
. As demonstrated by gp130 immunoprecipitation and
subsequent anti-phosphotyrosine immunoblotting, stimulation of
cardiomyocytes with CT-1, LIF, and a combination of IL-6 and sIL-6R
resulted in the rapid tyrosine phosphorylation of gp130. These data
indicate that tyrosine phosphorylation of gp130 is an early step in
CT-1 signaling, as it has previously been shown for the other members
of the IL-6 cytokine family(21, 22, 23) . As
determined by immunoblotting with an anti-phosphotyrosine antibody, LIF
induced the tyrosine phosphorylation of an additional
200 kDa
protein, which was not phosphorylated upon stimulation with the
IL-6/sIL-6R complex. Based on previous results, this protein most
likely corresponds to LIFR
(22, 26, 58) .
Stimulation of cardiac cells with CT-1 resulted in the tyrosine
phosphorylation of a protein, indistinguishable in size from LIFR
,
as well. Considering the ability of the LIFR
antagonist to block
the action of CT-1 in cardiomyocytes, the immunoblotting results
strongly suggest that CT-1, like LIF, induces the tyrosine
phosphorylation of LIFR
. In support of this conclusion, CT-1 has
recently been shown to bind to purified LIFR
in solution with
about the same affinity as LIF, and to interact with a cell surface
protein in M1 cells with a mobility expected for
LIFR
(54) . In summary, the present study indicates that
CT-1 signals through and induces tyrosine phosphorylation of the
gp130/LIFR
heterodimer in cardiomyocytes.
CT-1 Induces a Hypertrophic Phenotype in Cultured
Myocardial Cells Characterized by Sarcomere Assembly in Series and a
Selective Up-regulation of ANF Gene Expression
The present study
provides clear evidence that the CT-1 induced hypertrophic phenotype is
distinct from the hypertrophic phenotype observed following
G-protein-dependent stimulation. On a single cell level,
G-protein-dependent pathways induce a form of hypertrophy with a
relatively uniform increase in myocyte size and the addition of new
myofibrils in parallel(12, 15, 16) . By
contrast, as noted in the present study, the gp130/LIFR
-dependent
cytokines CT-1 and LIF induce an increase in myocyte size characterized
by a marked increase in cell length, but little or no change in cell
width. To characterize the effects of gp130/LIFR
-dependent
stimulation on the myofibrillar cytoarchitecture, cardiomyocytes were
dual-stained against thick (
MHC) and thin (F-actin) myofilaments,
and viewed by confocal laser microscopy(56) . Cardiomyocytes
stimulated with CT-1 and LIF displayed a high degree of myofibrillar
organization; myofibrils were organized in a strictly sarcomeric
pattern, were oriented along the longitudinal cell axis, and extended
to the cell periphery. Importantly, the increase in cell size and
length was associated with no change in the average sarcomere length,
strongly suggesting that the cell elongation in response to
gp130/LIFR
-dependent stimulation results from an addition of new
sarcomeric units in series. The morphologic changes induced by
gp130/LIFR
-dependent stimulation in vitro are reminiscent
of the changes observed in cardiac myocytes isolated from hearts
subjected to chronic volume overload(3, 4) . By
contrast, the pattern of cardiomyocyte hypertrophy induced by
-adrenergic stimulation more closely resembles a pressure
overload-like phenotype(1, 2) .On a molecular
level, gp130/LIFR
-dependent stimulation and
-adrenergic
stimulation resulted in distinct patterns of embryonic gene, MLC-2v,
and immediate early gene expression. Stimulation of cardiomyocytes with
CT-1 and LIF induced prepro-ANF mRNA expression, and perinuclear
accumulation and secretion of immunoreactive ANF. However, in contrast
to
-adrenergic stimulation, CT-1 and LIF did not induce skeletal
-actin expression. Growth factors, signaling through G-protein
coupled receptors induce ANF and skeletal
-actin in a coordinate
fashion(8, 11, 12, 13, 14) .
A recent study compared the expression pattern of distinct members of
the embryonic gene program in pressure overload versus volume
overload hypertrophy in vivo in the rat
myocardium(5) . As shown previously(52) , pressure
overload resulted in the coordinate induction of ANF and skeletal
-actin. However, volume overload hypertrophy was associated with a
selective increase in ANF expression, and no induction of skeletal
-actin, suggesting that the regulation of distinct embryonic genes in vivo is related to the hypertrophic stimulus(5) .
The pattern of embryonic gene expression induced by CT-1 and LIF in
cardiomyocyte culture therefore resembles the pattern observed in
volume overload hypertrophy in vivo.
Nuclear run-on
transcription assays revealed that the induction of prepro-ANF mRNA by
CT-1 and
-adrenergic stimulation is mediated, at least in part, by
an increased transcription of the ANF gene. The fact that the ANF mRNA
levels increased 5-fold, whereas the ANF gene transcription rate
increased only 2-fold, suggests that post-transcriptional mechanisms(s)
participate in the up-regulation of ANF gene expression in response to
CT-1 and PE(59) . In agreement with previous
results(11) , a 3003-bp ANF promoter fragment conferred
PE-inducible expression to a luciferase reporter gene. Likewise, the cis-regulatory elements mediating ANF inducibility in response
to endothelin 1 and
-thrombin reside within the proximal 3 kb of
the ANF 5`-flanking region, suggesting that G-protein-coupled receptor
agonists may utilize common signaling pathways for the induction of
ANF(12, 57) . By contrast, none of the
gp130/LIFR
-dependent cytokines (CT-1, LIF, and CNTF) activated the
3003-bp ANF promoter fragment. Considering the increased ANF gene
transcription rate in response to CT-1 and PE, we conclude that in
contrast to
-adrenergic stimulation, the CT-1 responsive cis-regulatory elements are located outside of the proximal 3
kb of the ANF 5`-flanking region. In recent studies, transgenic mice
harboring 3003 bp of ANF 5`-flanking region upstream of a luciferase
reporter gene were subjected to transverse aortic banding. Despite a
marked increase in left ventricular prepro-ANF mRNA expression, no
significant increase in reporter activity was observed(60) .
These findings demonstrate that the induction of ANF during cardiac
hypertrophy in vivo occurs through a distinct mechanism as
compared to G-protein-mediated cardiomyocyte hypertrophy in
vitro. Unraveling the mechanisms of ANF induction in response to
gp130/LIFR
-dependent stimulation may therefore provide insight
into the mechanisms governing the induction of ANF during in vivo cardiac hypertrophy.
CT-1 and LIF did not induce MLC-2v mRNA
expression, whereas phenylephrine stimulation resulted in a marginal
induction of MLC-2v. The induction of MLC-2v on the mRNA level may be a
feature unique to G-protein-coupled receptor stimulation of
cardiomyocytes in vitro(10, 12) : MLC-2v mRNA
expression is not up-regulated in the hypertrophic right ventricles of
pulmonary artery-banded mice (61) and in the hypertrophic
ventricles of transgenic mice overexpressing a constitutively active
-adrenergic receptor in the myocardium (62) .
A
distinct response to gp130/LIFR
-dependent versus
-adrenergic stimulation was also observed at the level of
immediate early gene induction. As compared to
-adrenergic
stimulation, CT-1 and LIF induced >10-fold higher levels of
c-myc and tis11 expression. Immediate early genes
encode known or putative transcription factors, and potential binding
sites have been identified in the promoter regions of a number of
cardiac genes (for review, see (63) ). The distinct
combinatorial expression of immediate early genes may therefore relate
to the differences in cell morphology and gene expression pattern in
response to gp130/LIFR
-dependent versus
-adrenergic
stimulation.
Taken together, these studies indicate that
gp130/LIFR
-dependent pathways can activate a hypertrophic
phenotype in cardiac muscle cells which is distinct both at a
morphological and a molecular level from the phenotype seen following
G-protein-dependent stimulation, and that is more consistent with a
volume overload, as opposed to pressure overload, hypertrophic
phenotype. Future studies in transgenic mice will be required to
rigorously test whether gp130/LIFR
-dependent signaling pathways
are implicated in volume overload and/or pressure overload ventricular
hypertrophy in vivo, in a manner analogous to recent studies
exploring the in vivo role of ras-dependent
pathways(18) . In this regard, double transgenic mice
overexpressing IL-6 and IL-6R have recently been
generated(64) . These mice display a constitutive tyrosine
phosphorylation (i.e. activation) of gp130 in the myocardium
and left ventricular hypertrophy with increases in cardiac weights and
cardiomyocyte volumes. These results suggest that the induction of
cardiomyocyte hypertrophy through gp130-dependent signaling pathways is
not confined to the in vitro hypertrophy assay, but may also
be observed in the in vivo context.
The question arises as
to the relative role of individual members of the IL-6 cytokine family
in the activation of a hypertrophic response in vivo. The
expression of IL-6R in the heart of wild type mice is extremely low,
making IL-6 an unlikely mediator of cardiac hypertrophy in
vivo. (
)Accordingly, a combination of IL-6 with sIL-6R
was required to induce tyrosine phosphorylation of gp130 in
cardiomyocytes in the present study. Likewise, the myocardium does not
express substantial levels of CNTFR
(30) , and
cardiomyocytes did not respond to CNTF stimulation in the present
study. It should be noted, however, that CNTF exerted marginal effects
on immediate early gene expression in cardiomyocytes. This might relate
to a weak interaction of CNTF with the gp130/LIFR
heterodimer in
the absence of CNTFR
(65, 66) . Alternatively, low
level expression of CNTFR
in cardiomyocytes may account for a weak
responsiveness to CNTF. The CT-1 mRNA is expressed in the heart from
adult mice(19) . RNase protection analysis of cardiac muscle
cells and cardiac-derived fibroblasts from neonatal rats reveals a
>10-fold higher level of CT-1 mRNA expression in cardiac muscle
cells versus cardiac fibroblasts, and CT-1 is expressed
primarily in cardiac muscle cells during murine
cardiogenesis(67) . By contrast, the expression of LIF mRNA in
the adult murine heart is extremely low as documented by RNase
protection analysis(68, 69) . These data would suggest
that CT-1 is a candidate cytokine activating gp130/LIFR
-dependent
signaling pathways within the myocardium, potentially through an
autocrine pathway. The generation of mice containing a null-mutation of
the CT-1 gene should provide a more definitive understanding of the in vivo role of CT-1 in development and in cardiac hypertrophy
in the adult animal.