(Received for publication, October 7, 1996, and in revised form, December 23, 1996)
From the Department of Medicine, § Center
for Molecular Genetics,
American Heart Association-Bugher
Foundation Center for Molecular Biology, and ¶¶ Department
of Pharmacology, University of California, San Diego, School of
Medicine, La Jolla, California 92093
Cardiac myocyte survival is of central importance in the maintenance of the function of heart, as well as in the development of a variety of cardiac diseases. To understand the molecular mechanisms that govern this function, we characterized apoptosis in cardiac muscle cells following serum deprivation. Cardiotrophin 1 (CT-1), a potent cardiac survival factor (Sheng, Z., Pennica, D., Wood, W. I., and Chien, K. R. (1996) Development (Camb.) 122, 419-428), is capable of inhibiting apoptosis in cardiac myocytes. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 activated both signal transducer and activator of transcription 3 (STAT3)- and mitogen-activated protein (MAP) kinase-dependent pathways. The transfection of a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into myocardial cells blocked the antiapoptotic effects of CT-1, indicating a requirement of the MAP kinase pathway for the survival effect of CT-1. A MEK-specific inhibitor (PD098059) (Dudley, D. T., Pang, L., Decker, S.-J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci. USA 92, 7686-7689) is capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. In contrast, this inhibitor did not block the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Therefore, CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires MAP kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase.
Cardiac muscle cell survival plays a critical role in maintaining the normal function of the heart and possibly in cardiac development. Adult cardiac muscle cells are terminally differentiated and therefore have lost their proliferative capacity. In contrast to skeletal muscle, the myocardium does not contain satellite heart muscle cells, and irreversible heart injury results in scarring and an eventual decrease in global cardiac function. In response to mechanical stimuli and hemodynamic stress, the adult myocardium activates an adaptive hypertrophic response that is characterized by an increase in myocardial cell size without a concomitant increase in myocyte number (For review, see Refs. 1 and 2). However, during long-standing exposure to hypertension or other forms of hemodynamic stress, a distinct form of myocardial cell hypertrophy can be activated in which the heart becomes dilated and individual cardiac myocytes exhibit an increase in cell length, reflecting the addition of new sarcomeric units in series (3, 4). This dilatation of the heart is usually accompanied by fibrosis, microscarring, and the loss of viable cardiac myocytes throughout the myocardium. As a result of cardiac dilatation and myocyte dropout, the myocardium ultimately develops an irreversible loss of function and ensuing cardiac muscle failure (4). As such, the identification of the signaling pathways that mediate distinct forms of cardiac muscle cell hypertrophy, dysfunction, and cardiac muscle cell survival are critical to the ultimate elucidation of the molecular basis of cardiac muscle failure.
By coupling expression cloning with an embryonic stem cell-based model
of in vitro cardiogenesis (5), recent studies have identified cardiotrophin 1 (CT-1),1 a novel
cardiac cytokine that was isolated in a search for new factors that
induce cardiac myocyte hypertrophy (5). CT-1 is a new member of the
IL-6 family of cytokines that exert their biological effects through
the shared signaling subunit gp130 (3, 5-7) and can activate a
distinct form of myocardial cell hypertrophy that is characteristic of
volume overload cardiac hypertrophy at the molecular, morphological,
and cellular levels (3). Importantly, cardiotrophin 1 has been shown to
be capable of promoting survival of both embryonic and neonatal rat
ventricular muscle cells (8). Recent studies have demonstrated that
CT-1 exerts its effects on cardiac muscle cell hypertrophy through promoting the heterodimerization of gp130 with the leukemia inhibitory factor (LIF) receptor , both of which are required for the
activation of the downstream hypertrophic response (3). Relatively less is known concerning the mechanisms by which CT-1 promotes cardiac myocyte survival. It is unclear whether this effect is based on a
generalized trophic effect or a specific requirement for CT-1 for
long-term myocyte survival or whether it reflects the activation of
signaling pathways that can act to block programmed cell death of
cardiac myocytes, i.e. apoptosis. In addition, it is unknown whether divergent or convergent downstream signaling pathways mediate
these two distinct effects of CT-1 on myocyte survival and hypertrophic
responses.
To address these questions, the current study reports the characterization of an in vitro cardiac muscle assay system in which apoptosis is induced following serum deprivation of myocytes that are plated at a relatively low density. In this assay system, we document the onset of cardiac myocyte cell death via apoptotic pathways by two independent criteria, i.e. scoring for nuclear changes associated with apoptosis and the presence of internucleosomal DNA fragmentation. The addition of CT-1 is capable of promoting cardiac myocyte survival and blocking apoptosis. To explore the potential downstream pathways that might be responsible for this effect, we documented that CT-1 is capable of activating both STAT3- and MAP kinase-dependent pathways. To directly relate the activation of these pathways to the biological functions of CT-1, we transfected a MAP kinase kinase 1 (MEK1) dominant negative mutant cDNA into neonatal ventricular myocardial cells and found that the mutant was capable of blocking the antiapoptotic effects of CT-1 on individual cardiac myocytes, thereby indicating a requirement of MAP kinase activity for the survival effect of CT-1 on cardiac myocytes. In addition, in studies applying the MEK inhibitor (PD098059) (9), we observed that the inhibitor was capable of blocking the activation of MAP kinase, as well as the survival effect of CT-1. The inhibitor displayed specificity for the MAP kinase pathway, as it did not inhibit the activation of STAT3, nor did it have any effect on the hypertrophic response elicited following stimulation of CT-1. Taken together, these studies indicate that CT-1 promotes cardiac myocyte survival by preventing apoptosis through a signaling pathway that requires MAP kinase. In addition, MAP kinase does not appear to be required for the activation of a CT-1-dependent hypertrophic response, indicating that CT-1 uses divergent signaling pathways for the activation of the survival and hypertrophic responses, the latter of which may be mediated by a JAK/STAT or MEK kinase/c-Jun NH2-terminal protein kinase pathway.
Murine LIF, IL-6, and ciliary neurotrophic factor (CNTF) were obtained from Life Technologies, Inc., Genzyme, and Boehringer Mannheim, respectively. Mouse CT-1 was a kind gift from Diane Pennica (Genentech Inc., South San Francisco, CA); STAT3 monoclonal antibodies were purchased from Upstate Biotechnology Inc.; ERK1 and ERK2 monoclonal antibodies were obtained from Transduction Laboratories. PD098059 was a kind gift from A. R. Saltiel (Warner Lambert Co., Ann Arbor, MI).
Cell CulturesRat neonatal cardiac ventricular myocytes were prepared as described previously (8). After the cardiac myocytes were purified by Percoll gradient, the cells were preplated on a noncoated dish for 1 h twice to reduce contaminating cardiac fibroblasts, which often constitute 5% of the purified cardiac myocytes. The cells were plated in defined medium containing 10 mM glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin in Dulbecco's modified Eagle's medium in a plate coated with 1% gelatin. After 4 days of culture, the cells were then immunostained with rabbit anti-rat ventricular myosin light chain 2 (MLC-2v) antibody, as described previously (10). More than 95% of the cells displayed positive MLC-2v staining.
Northern BlotTotal RNA from neonatal rat cardiac myocytes
was isolated by using a RNAzol solution (Cinna/Biotecx), according to
suggested conditions of the manufacturer. A rat ANF full-length
cDNA probe was labeled with [32P]dCTP. 5 µg of
total RNA was run on a 1% agarose gel and transferred to a nylon
membrane. Prehybridization and hybridization were performed by using a
quick hybridization solution (Stratagene). After washing, the filter
was exposed to x-ray film at 70 °C for 3 h. The filter was
then stripped and hybridized with a glyceraldehyde-3-phosphate dehydrogenase probe and exposed to a film at
70 °C for 3 h.
Purified rat neonatal myocytes were plated in triplicate in a 96-well microplate with 1.2 × 104 cells/well with the plating medium and maintained at 10% CO2 and 37 °C for 24 h. The cells were then washed twice with the above medium in the absence of serum, followed by the addition of 1 nM CT-1 to the wells. The cells were stained with 3-(4,5-dimethyl thiaziazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) and counted as described previously (8). All of the experiments were performed in triplicate and repeated at least three times.
MAP Kinase AssayThis assay was performed as described
previously (11) with slight modification. After plating, cardiac
myocytes (1 × 106 cells/6 cm) were washed and
maintained in serum-free medium for 24 h prior to experimentation.
Cells were then treated with CT-1, LIF, and IL-6 (1 nM, the
concentration that was able to fully activate the kinases) for 12 min,
washed with cold phosphate-buffered saline, and lysed in Tris-buffered
saline containing 1% Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 500 µM Na3VO4, and 1 mM NaP2. The cell lysates were carefully
collected and taken for protein determination (Bio-Rad). Equal amounts
of lysate were incubated with ERK1 and ERK2 antibody (Transduction
Laboratories) and protein A-Sepharose CL-4B (Pharmacia Biotech Inc.)
for 1 h at 4 °C. The precipitate samples were then diluted with
a kinase buffer containing 20 mM Hepes, pH 7.0, 2 µM dithiothreitol, 20 mM ATP, 10 mM MgCl2, 4 µCi of
[-32P]ATP, and 30 µg of myelin basic protein. After
incubation at room temperature for 30 min and boiling for 5 min, equal
samples were loaded onto 15% SDS-polyacrylamide gel electrophoresis.
The gel was dried and exposed to Kodak x-ray film at 70 °C for
3 h. All of the assays were repeated two to three times.
Purified neonatal rat cardiac myocytes (2 × 106 cells/10 cm) were cultured in serum-free medium in the presence or absence of cytokines for various periods. The cells were then washed with cold PBS, and the DNA was collected by using a DNA isolation kit (Purogene). 2 µg of DNA was loaded onto a 2% agarose gel. TUNEL assays were performed after the cells were fixed with 4% paraformaldehyde for 30 min at 25 °C and washed with PBS three times. Terminal deoxynucleotidyl transferase reaction solution containing 0.2 M potassium carodylate pH 7.2, 4 mM MgCl, 1 mM 2-mercaptoethanol, and 0.5 mg/ml bovine serum albumin was used to equilibrate the cells. Terminal deoxynucleotidyl transferase reaction solution containing 2 mM biotin-conjugated dUTP (Boehringer Mannheim) and 10 units of terminal deoxynucleotidyl transferase (Life Technologies) was added to the cells for 60 min in a 37 °C humidified incubator. After washing with PBS containing 0.2% Tween 20, the cells were incubated with a fluorescein isothiocyanate-tagged anti-biotin monoclonal antibody (Jackson Laboratory) (1:500) for 1 h at 37 °C. After washing with PBS containing 0.2% Tween 20, the cells were then imaged with fluorescent microscopy.
Transfection and ImmunostainingFor calcium phosphate
transfections, purified cardiac myocytes were exposed to a
cDNA-calcium phosphate precipitate 24 h after plating (11). A
firefly luciferase cDNA controlled by a Rous sarcoma virus promoter
and pcDNA3 backbone vector (Invitrogen) or pcDNA3 containing
the MEK1 K97M mutant (12) (a kind gift from Natalie Ahn, University of
Colorado, Boulder, CO) were co-transfected. Following transfection, the
cells were washed and maintained in serum-free medium in the presence
of CT-1 (1 nM). The cells were then incubated for 4 days
and fixed. Immunostaining was performed as described previously (10).
MLC-2v antibody or -myosin heavyweight chain monoclonal antibody (a
kind gift from Jim Lin, University of Iowa, Iowa City, IA) were used to
identify cardiac myocytes; an anti-luciferase antibody (Cortex) was
used to identify successfully transfected cells. Hoescht dye was used
to stain nuclei to identify apoptosis.
After serum starving for 24 h, cardiac myocytes (4 × 106/10 cm) were treated with agonists (1 nM) and lysed with a solution containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, and 0.5% Nonidet P-40. Cell lysates (150 µg of protein, 200 µl) were incubated with anti-STAT3 mouse monoclonal antibody (Transduction Laboratories) at 4 °C for 12-18 h and with 20 µl of 50% suspension of anti-mouse IgG-agarose (Sigma) for an additional 30 min. The precipitate was extracted and subjected to Western blotting analysis using anti-STAT3 and horseradish peroxidase-conjugated anti-phosphotyrosine antibodies (Transduction Laboratories) following the manufacturer's protocol.
Statistical AnalysisData are presented as means ± S.E. p values were determined using one way analysis of variance.
Previous studies have established a loss of
cardiac myocyte survival in short-term culture in the absence of serum
(8). To investigate whether cardiac myocyte death after serum
deprivation is secondary to apoptosis, we first evaluated cells for the
presence of internucleosomal cleavage by monitoring for DNA laddering, a hallmark of apoptosis, on agarose gels. The DNA fragmentation of
cultured neonatal ventricular cells was observed after day 2 of serum
deprivation and persisted throughout day 6 in the present assay system
(Fig. 1A), whereas little DNA laddering was
observed in the presence of serum (data not shown). DNA fragmentation
cleavage was not present in noncardiac cells even after 10 days in
serum-free media (Fig. 1C), documenting that the fragment
DNA did not come from the small proportion (5%) of contaminating
nonmuscle cells in the culture. Cells undergoing apoptosis were
also identified at the single cell level by TUNEL staining and by
nuclear staining with the Hoescht 33258 dye (Fig. 2).
About 70% of myocytes maintained in serum-free media for 5 days were
TUNEL stained positive and displayed small condensed nuclei, cell
shrinkage, and nuclear fragmentation consistent with apoptosis (Fig.
2). Thus, it is clear that cardiac myocytes in the absence of serum
displayed several independent features of apoptosis, such as cell
shrinkage, chromosomal DNA condensation, and fragmentation (Fig. 2,
D-F). These results indicate that, similar to other cell
types such as lymphocytes and neural cells, cardiac myocytes also
undergo apoptosis in the absence of defined growth factors.
CT-1 Inhibits Apoptosis in Serum-deprived Neonatal Rat Myocytes
To determine whether the effects of CT-1 on promoting
embryonic and neonatal cardiac survival (8) are associated with
inhibition of cardiac myocyte apoptosis, we examined the effects of
CT-1 in cultured cardiac myocytes. As shown in Fig. 1B, we
found that 1 nM CT-1 inhibited internucleosomal cleavage of
genomic DNA in serum-deprived cardiac myocytes. This inhibition
occurred in a concentration-dependent manner (data not
shown). The antiapoptotic effect of CT-1 was further revealed by TUNEL
staining and by staining the nuclei with Hoescht 33258 dye (Fig. 2,
A-C). Fewer CT-1-treated cells (less than 10%) were
positive for internucleosomal cleavage by TUNEL staining. In addition,
CT-1-treated cells did not display chromatin condensation and cell
shrinkage that is characteristic of myocyte apoptosis. LIF and
insulin-like growth factor 1 were also effective in preventing
apoptosis in cardiac myocytes, whereas IL-6, CNTF, basic or acidic
fibroblast growth factors (100 nM), and transforming growth
factor (10 nM) failed to inhibit apoptosis (Fig.
1B; data not shown). Therefore, these results indicate that CT-1 is a potent survival factor for preventing myocyte apoptosis.
Previous
studies have documented that IL-6 could activate MAP kinase via gp130
(7, 13). To determine whether CT-1 can also activate MAP kinases in
cardiac myocytes, neonatal ventricular muscle cells maintained in
serum-free media were stimulated with CT-1 and other cytokines for
various periods. Cellular lysates were then purified, and two forms of
MAP kinase present in cardiac myocytes (ERK1 or ERK2) were
immunoprecipitated using specific ERK1 and ERK2 antibodies. MAP kinase
activity was subsequently determined by measuring its ability to
phosphorylate (myelin basic protein). CT-1 activated both ERK1 and ERK2
by 4-5-fold at 10-15 min after stimulation (Fig. 3).
This activity decreased to basal level after 30 min (data not shown),
similar to the time course that was seen following phenylephrine
stimulation of cardiac myocytes (11). In addition, Western blotting of
immunoprecipitated proteins by ERK2 antibody revealed that CT-1 could
induce the increase of the phosphorylation form of ERK2 protein (data
not shown). In comparison with other cytokines, we found that LIF also
activated MAP kinase activity (Fig. 3), but IL-6 had less of an effect
in some experiments. Since IL-6 did not display detectable biological effects on cardiac myocyte proliferation, hypertrophy, and survival, or
on the phosphorylation of gp130 and STAT3 (3, 8, Fig. 8), the low
activation of MAP kinase most likely represents a signal derived from
the effects of IL-6 on the 5% noncardiac cells that are present in the
cultures. Thus, these results demonstrated that both CT-1 and LIF can
activate MAP kinase pathways in cardiac myocytes.
MEK1 Dominant Negative Mutant Blocks the Survival Effects of CT-1
To determine whether the activation of MAP kinase is
required for CT-1 inhibition of apoptosis in cardiac myocytes, we
transiently expressed the dominant negative mutant of MEK1 (K97M) in
cardiac myocytes. This mutant protein can be phosphorylated but does
not activate the downstream kinases (12). The MEK1 (K97M) expression vector was co-transfected with Rous sarcoma virus luciferase into neonatal rat cardiac myocytes. The cells were then triple immunostained with an anti-luciferase antibody for identifying the transfected cells,
an anti--myosin heavyweight chain monoclonal antibody for
identifying cardiac myocytes, and Hoescht 33258 dye for identifying cells that were undergoing apoptosis. 27% of myocytes transfected with
MEK1 (K97M) were apoptotic even in the presence of CT-1. However, only
6% of the myocytes transfected with the luciferase vector or empty
backbone vector were apoptotic in the presence of CT-1 (Fig.
4). These results were independently confirmed by TUNEL
staining, in which 23% of cells transfected with MEK1 (K97M) and
stimulated with CT-1 were positive for apoptosis, compared with 5% in
control groups (data not shown). Since transfection efficiency in
cardiac myocytes is generally low (about 1-2% of cells), it is
difficult to confirm using other assays, such as DNA laddering on
agarose gels, that the MEK1 dominant negative mutant can inhibit
antiapoptotic effects of CT-1. However, the results from the above
assays are consistent with the results with a MEK inhibitor (see
below). These data suggested that activation of a MAP
kinase-dependent pathway is required for the survival effects of CT-1 on cardiac myocytes.
PD098059 Inhibited the Activation of MAP Kinases and Survival Effects of CT-1
Although the application of dominant negative
mutants provides useful information for dissecting signaling pathways,
it has limitations that include the low transfection efficiency in
certain cell types and unwanted interaction of the mutants with other components. Small cell-permeable molecules, such as inhibitors for
intracellular serine and threonine kinases, provide an alternative approach (9). PD098059 is a specific MEK inhibitor that selectively inhibits MEK1 activity (9, 14). This inhibitor blocks phosphorylation and activation of MAP kinase-induced growth factors (9, 14, 15). For
example, nerve growth factor-induced differentiation of PC-12 cells was
blocked effectively by this agent. To confirm that activation of MAP
kinase is required for CT-1-stimulated inhibition of apoptosis in
cardiac myocytes, we used PD098059 in our experimental system to
determine whether this agent could inhibit the activation of the ERK1
and ERK2 kinases concomitantly with relieving the CT-1 inhibition of
myocyte apoptosis. Similar to results in other cell systems, PD098059
inhibited the activation of both ERK1 and ERK2 in cardiac myocytes in a
concentration-dependent fashion (Fig. 5). At
1 µM PD098059 a partial inhibition and at 10 µM a complete inhibition of CT-1-stimulated activation of
ERK1 and ERK2 were observed. PD098059 had an almost identical
inhibitory effect on these kinases in cardiac myocytes stimulated by
phenylephrine (11). This inhibitor was then used to determine whether
activation of MAP kinase by CT-1 was required for cardiac myocyte
survival. As shown in Fig. 6, 10 µM
PD098059 was able to completely inhibit the survival effects of 1 nM CT-1. Increasing the concentration of CT-1 to 100 nM was not able to prevent apoptosis in the presence of 10 µM PD098059. This result suggested that PD098059 might
block the downstream signaling pathway by which CT-1 prevents
apoptosis. It is notable that the survival effects of CT-1 in the
current study are relatively higher than previously reported (8). The difference between the current and previous study is the cell density
that was used in the in vitro assay system. It is clear that
a higher cell density provides a more suitable condition for cardiac
myocyte survival. Although the conclusions regarding the protective
effects of CT-1 on both lower and higher cell density cultures are
qualitatively identical, we believe that the currently modified
experimental conditions constitute a more optimal assay system for
determining the survival effects of growth factors on cardiac myocytes.
To exclude the possibility of a nonspecific cytotoxic effect of
PD098059, we tested whether it was capable of inducing cell death in
the presence of serum. 5% fetal bovine serum was added to the myocytes
after the addition of PD098059. Serum-induced survival was not
inhibited by 10 or 100 µM PD098059 (Fig. 6B;
data not shown). This result is in agreement with previous studies that
showed that PD098059 has no obvious nonspecific cytotoxic effects on
PC-12 cell lines (15). This result suggests that PD098059 may induce
cell death by specifically inhibiting the MAP kinase pathway and not
through a nonspecific cytotoxic effect on cardiac myocytes. Taken
together, these results provided additional evidence that the
activation of the MAP kinase pathway is required for the survival
effects of CT-1 on cardiac myocytes.
PD098059 Does Not Inhibit CT-1-stimulated Hypertrophy or Induction of ANF
Since CT-1 also promotes cardiac myocyte hypertrophy (5),
it is important to determine whether shared or divergent signaling pathways mediate the survival and hypertrophic effects of CT-1 on
cardiac myocytes. One of the advantages of the in vitro
hypertrophy model is that cultured cardiac myocytes can quickly respond
to many stimuli that can induce morphological, biochemical, and
molecular changes characteristic of hypertrophy. To determine whether
CT-1 could stimulate hypertrophy in the presence of PD098059, we
evaluated the changes in myocyte morphology and ANF gene expression.
CT-1 stimulated an increase in cell size and an increase in ANF
mRNA within 1-3 days after stimulation. This period precedes the
onset of significant myocyte cell death in the presence of PD098059. Even though 10 µM PD098059 effectively inhibited the
effect of CT-1 on the activation of MAP kinase and cell survival, it
had little effect on the approximate 4-fold increase of ANF mRNA
stimulated by CT-1 at 48 h after stimulation (Fig.
7A). Thus, PD098059 did not significantly
affect ANF expression induced by CT-1 (1 nM). As reported
previously, CT-1 induces a form of hypertrophy characterized by
elongation of cardiac myocytes and the assembly of new sarcomeric units
in series (3, 5). PD098059 was not able to block the ability of CT-1 to
induce these morphological changes, even at day 4, when PD098059
induced a significant amount of cell death (Fig. 7B). These
findings suggested that CT-1 activation of myocyte hypertrophy does not
require MAP kinase signaling.
PD098059 Does Not Block the Activation of the STAT3 Pathway by CT-1
The findings described above suggest that there is a MAP kinase-independent signaling pathway that may mediate the CT-1-stimulated myocyte hypertrophy. Recently, constitutive activation of gp130 by the overexpression of both IL-6 and IL-6 receptor in the hearts of transgenic mice was shown to be associated with ventricular hypertrophy and increased JAK/STAT3 activity (16). Thus, we sought to determine whether CT-1 is also able to activate STAT3 kinase. Stimulation of ventricular myocytes with both CT-1 and LIF increased phosphorylation of STAT3, whereas stimulation with IL-6 had no effect on STAT3 phosphorylation. To determine whether the activation of STAT3 by CT-1 required activation of MAP kinase, we incubated ventricular myocytes in the presence of CT-1 and 10 µM PD098059. Inhibition of MAP kinase activity was not able to prevent phosphorylation of STAT3, suggesting that CT-1 can activate both MAP kinase and MAP kinase-independent signaling cascades (Fig. 8B). These data also indicate that STAT3 activation is not sufficient to maintain cardiac myocyte survival when MAP kinase is inhibited by PD098059. It remains possible that JAK/STAT3 may be needed for CT-1-induced cardiac hypertrophy. In addition, these studies suggest the simultaneous activation of both JAK/STAT and MAP kinase pathways by CT-1 in cardiac muscle cells and may provide a useful system to study the differential regulation of signaling pathways for survival versus hypertrophy in cardiac myocytes.
Adult cardiac muscle
cells are terminally differentiated and have lost their proliferative
capacity. As a result, the maintenance of cardiac muscle cell survival
is critical for the maintenance of normal cardiac function. Although a
wide variety of survival factors have been found for neuronal cells and
several other terminally differentiated cell types (17-19), and
several growth factors may play important roles in cardiac hypertrophy
and cardiac development (20), relatively little is known about the
specific combination of growth factors and/or cytokines that are
required to maintain long-term survival of cardiac myocytes. In
neuronal cell types, a number of peripherally derived neurotrophic
factors have been shown to play a vital role in the regulation of the
survival of spinal motoneurons at a variety of stages of development
(21). In this regard, two members of the IL-6 family of cytokines, LIF and CNTF, have been shown to play an important role in maintaining the
viability of motoneurons in long-term culture and also in the in
vivo context (21-25). Interestingly, mice that harbor a targeted
disruption of the subunit of the CNTF receptor or the
receptor
of the LIF receptor die shortly after birth and display a severe loss
of more than 48% of the motoneurons in the spinal cord and brain stem
(26, 27). These results suggest that ligands that activate gp130 may
play a critical physiological role in regulating survival of terminally
differentiated cell types in vivo. However, the function of
the growth factors or cytokines in cardiac myocyte survival and its
signaling pathways by which this survival is conferred remain
unclear.
Recently, CT-1 has been identified and cloned based on its ability to induce hypertrophy in cultured neonatal rat ventricular muscle cells (5). CT-1 is expressed relatively early during murine cardiogenesis (E8.5), at which time it displays preferential expression in cardiac muscle, with little expression found in the mesenchymally derived atrioventricular cushions and conotruncal ridges, which contribute to septation of the chambers and outflow tract, respectively (8). In later stages, cardiotrophin 1 is found in a wide variety of other tissues, including neuronal cells, dorsal root ganglion, and skeletal muscle (8). Functionally, CT-1 promotes in vitro cultured cardiac myocyte survival (8). Recently, cardiotrophin 1 has also been shown to display both in vitro and in vivo effects on survival of neonatal motoneurons following sciatic nerve axotomy (28). However, the mechanism underlying the survival effects of CT-1 remains unclear.
In this regard, the present study provides direct evidence that one of
the mechanisms by which cardiotrophin 1 can promote the survival of
terminally differentiated cell types is via the activation of pathways
that ultimately lead to inhibition of the apoptotic signaling pathway.
The present study characterizes an in vitro assay system
whereby cardiac myocytes enter the apoptotic signaling pathway after
the deprivation of serum. Using three independent criteria, TUNEL
staining, internucleosomal DNA fragmentation, and nuclear condensation,
we have documented that CT-1 can block the onset of apoptosis in
individual cardiac muscle cells. This effect appears to be specific and
does not simply represent an indirect trophic effect on the cells, as
agents such as transforming growth factor , which has been shown to
have trophic effects on cultured neonatal rat ventricular muscle cells
(20), are without significant effect. Finally, the addition of IL-6 or
CNTF has relatively little effect, whereas the addition of another member of the IL-6 family (LIF), which uses an identical heterodimer pathway for activating downstream cardiac muscle cell responses via
gp130 and LIF receptor
(3), has a similar effect on the blockade of
the apoptotic signaling pathway. Insulin-like growth factor 1, which
has been shown to be cardioprotective in a murine model of myocardial
ischemia reperfusion (29), could also inhibit cardiac myocyte
apoptosis. Taken together, these studies provide the first evidence
that cardiotrophin 1 promotes cardiac myocyte survival through the
activation of pathways that can interrupt the apoptotic signaling
cascade. Recently, we also found that CT-1 could block the apoptosis in
neonatal cardiac myocytes infected with Coxsackie
virus.2 The effects of CT-1 on adult heart
cells are currently under study.
All members of the IL-6-LIF cytokine family trigger
downstream signaling pathways in multiple cell types through the
homodimerization of gp130 or the heterodimerization of gp130 and LIF
receptor (7). Until now, intracellular signaling pathways that
couple the gp130 activation with the downstream cardiac cell responses have remained unclear. In other cell types, members of this family have
been shown to activate the JAK/STAT pathway (7, 30-32). STAT3 is
phosphorylated in response to IL-6-related cytokines and plays a
critical role in gp130-mediated terminal differentiation and growth
arrest of a myeloid cell line (32-34). It is also becoming clear that
this family of cytokines can activate Ras and MAP kinase cascades, as
well (7, 13). However, the distinctive role of this Ras/MAP kinase
pathway versus the JAK/STAT pathway in the activation of
downstream cellular responses of the IL-6 family is not completely
clear.
Using two independent approaches, the current study provides direct evidence that MAP kinase-dependent pathways are required for the inhibition of cardiac myocyte apoptosis. Transfection studies with the MEK1 dominant negative mutant protein vector result in blockade of the CT-1 inhibition of myocyte apoptosis. To confirm the essential role of the MAP kinase pathway in the survival function of CT-1, we applied a MEK-specific inhibitor, PD098059, in our assay system. In addition, PD098059 prevents MEK1 activation by Raf and has been shown to have little effect on other kinases, including cAMP-dependent kinase, protein kinase C, and other serine and threonine kinases (9, 14, 15). This inhibitor does not inhibit c-Jun NH2-terminal protein kinase activation either.3 In the current study, PD098059 effectively inhibited the activation of ERK1 and ERK2 following CT-1 stimulation in a concentrationdependent manner but had little effect on activation of the JAK/STAT pathway, as assessed by the phosphorylation of STAT3, thereby providing further evidence for selectivity of these inhibitory effects on MAP kinase. In addition, treatment with this inhibitor significantly blocked the survival effects of CT-1 and led to an increase in the loss of cell viability. The time course of the onset of cell death induced by the inhibitor was similar to the time course found during serum deprivation, thereby suggesting that cell death may result from similar pathways. The fact that the effects of PD098059 on the cells cannot be rescued by higher concentrations of CT-1 suggested that PD098059 is selectively targeting downstream CT-1-dependent signaling pathways.
Recently, MAP kinase pathways have also been found to be necessary for nerve growth factor effects on promoting the survival of neuronal cell types (PC-12), whereas c-Jun NH2-terminal protein kinase activation and inhibition of MAP kinase has been shown to be critical for induction of apoptosis in these cells (35). The current study provides further evidence that MAP kinase-dependent pathways might play a particularly important role in promoting the survival of terminally differentiated cell types. However, it should be noted that MAP kinase-independent pathways for the inhibition of cardiac myocyte apoptosis also exist, since the current studies document that serum can block the apoptosis of cardiac myocytes in this cultured assay system even in the presence of the selective MAP kinase inhibitor. It will become of interest to determine whether similar MAP kinase-dependent signaling pathways operate in the in vivo context and to identify the downstream cellular effectors of this inhibition. In this regard, recent studies have suggested that the induction of B-cell lymphoma/leukemia X and 2 can result in the inhibition of apoptosis in a wide variety of cell types (36, 37). It will certainly become of interest to determine whether this induction is dependent on MAP kinase and whether this is sufficient to confer a protective effect on myocardial cells.
CT-1-dependent Hypertrophy Is Independent of MAP KinaseIn addition to its effects on promoting cardiac myocyte
survival, CT-1 is also capable of activating a distinct form of
myocardial cell hypertrophy in a well characterized in vitro
assay system (3). The overexpression of both IL-6 and IL-6 receptors in the heart of transgenic mice results in cardiac hypertrophy, which is
associated with constitutive activation of STAT3 activity (16). In
addition to gp130-dependent pathways for cardiac myocyte
hypertrophy, it has been well documented that G protein-coupled
receptors, including -adrenergic and endothelin 1 receptor subtypes,
can activate features of myocardial cell hypertrophy in an in
vitro assay system (38-41). Moreover, Ras-dependent
pathways appear to be both necessary and sufficient to activate
hypertrophy in vitro and in vivo, as recently
revealed by transgenic mice that express constitutively active Ras in
the ventricular chamber under the control of the MLC-2v promoter (42).
A substrain of these mice display a distinct form of obstructive
cardiac hypertrophy4 indistinguishable at a
morphological, physiological, and pathological level from hypertrophic
cardiomyopathy in the clinical setting. Interestingly, the phenotype
seen in CT-1-stimulated cells correlates mostly with the volume
overload hypertrophy phenotype, resulting in the addition of sarcomeric
units in series as opposed to in parallel (3). In addition, divergent
signaling pathways appear to mediate the activation of these distinct
forms of hypertrophy in this neonatal assay system. Thus, although
Ras-dependent pathways appear to be sufficient for
activating hypertrophy in the system, it is not clear that MAP kinase
is the main downstream effector for this response. In fact, recent
studies have shown conflicting results regarding the requirement of MAP
kinase pathways in hypertrophy induced by
-adrenergic agonists (11,
43, 44).
In the present study, although we document that the MAP kinase pathway
is required for CT-1-dependent effects on the inhibition of
cardiac myocyte apoptosis, it also appears that MAP kinase is not
required for the activation of morphological features of hypertrophy
and the concomitant induction of an embryonic marker of the response,
the ANF gene, by CT-1. These results are consistent with recent studies
that have also suggested that MAP kinase is not sufficient for the
activation of hypertrophy following G protein-coupled receptor-dependent pathways (11). Previous studies have
suggested that MAP kinase activity may be involved in -adrenergic-
or endothelin 1-induced hypertrophy (43, 44). The possibility exists
that multiple signaling pathways may be required to activate cardiac hypertrophy, and that a single downstream MAP kinase pathway might be
insufficient to trigger a hypertrophic response. In this regard, our
results indicate that other signaling pathways may be responsible for
CT-1-induced hypertrophy, whereas MAP kinase activity may not be
essential. In addition, these data support the notion that divergent
signaling pathways mediate the distinct effects of CT-1 on inhibition
of cardiac myocyte apoptosis and the activation of cardiac myocyte
hypertrophy. Although cross-talk between JAK/STAT and MAP kinase
pathways has been demonstrated in certain cell types (45), in the
current study it appears that the inhibition of MAP
kinase-dependent pathways does not dramatically effect the
phosphorylation of STAT3, suggesting that the cross-talk between these
two pathways may be dependent on the specific cytokine and particular
cell context. Taken together, our results suggest that the STAT3
pathway may not be sufficient to mediate cardiac myocyte survival but
may be involved in the hypertrophy promoted by CT-1.
Based on the results of the present and previous studies (7, 16,
30-33), we have proposed a working model of the downstream signaling
pathways that may mediate the actions of CT-1 on cardiac muscle cells
(Fig. 9). CT-1 activates downstream responses through promoting the heterodimerization of the LIF receptor and gp130 and
subsequently activates JAK. The latter activates both STAT3- and MAP
kinase-dependent pathways. MAP kinase-dependent
pathways appear to be responsible for promoting the survival effects of CT-1 through the activation of pathways that ultimately inhibit cardiac
myocyte apoptosis. The question arises of whether this is secondary to
the induction of endogenous apoptotic inhibitory pathways. On the other
hand, the constitutive activation of STAT pathways, which are
associated with hypertrophy in the setting of IL-6 and IL-6 receptor
transgenic mice, have been implicated in the activation of hypertrophic
effects of gp130 stimulation in the in vivo context (16).
Future studies will be designed to directly test whether these
divergent pathways promote cardiac myocyte survival and hypertrophy
through these distinct pathways and to determine whether there is
cross-talk with other pathways that also might lie downstream of the
CT-1 stimulation, such as c-Jun NH2-terminal protein kinase
activation. Given the ability to resolve specific cardiac phenotypes
following stimulation of cardiac muscle cells with defined agonists,
CT-1 stimulation of cardiac muscle cells may serve as a valid system to
identify the nodal points in the signaling pathway that mediate the
activation of these two important responses of cardiac muscle cells on
survival and growth responses. Experiments using genetically engineered mouse model systems harboring selective activation of these constituent pathways in ventricular muscle cells, coupled with miniaturized physiological technology to discriminate these distinct phenotypes, are
currently in progress to directly test the validity of this model.
We gratefully acknowledge the assistance of Judy Brundrett in the preparation of the manuscript.