From the Departments of Biochemistry and Molecular
Biology and ¶ Internal Medicine, The University of Texas,
Houston Medical School, Houston, Texas 77030
Received for publication, August 20, 2002, and in revised form, December 10, 2002
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ABSTRACT |
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The addition of glutamine as a major
nutrient to cultured neonatal rat cardiomyocytes produced an increase
in myocyte size and the organization of actin into myofibrillar arrays.
The cellular response was associated with increased abundance of the
mRNAs encoding the contractile proteins, Cardiac hypertrophy is an adaptive process accompanied with a
series of changes in gene expression allowing the heart to maintain or
increase cardiac output in response to increased workload (1-3). Physiological hypertrophy refers to the enlargement of the heart resulting from repeated endurance exercise. This form of cardiac hypertrophy is both beneficial and reversible. It is characterized by
increased total RNA and total protein with increased abundance of the
adult contractile isoforms, In recent years, efforts to identify signaling pathways associated with
cardiac hypertrophy have largely focused on the role of calcium
signaling in mediating the cardiac hypertrophic response (12). In this
regard the calcineurin-NFAT signaling pathway has received the most
attention (12, 13). However, in eukaryotic cells one of the most
important intracellular transducers of growth-related signaling is the
rapamycin-sensitive pathway associated with mammalian target of
rapamycin (mTOR), a phosphatidylinositol kinase-related protein kinase
that is regulated in response to growth factors and nutritional status
(14). mTOR functions at a nexus of protein kinase signaling,
receiving input from numerous upstream signaling pathways and
delivering instructions to a variety of downstream effectors
controlling the cellular response to mitogenic stimuli (15). mTOR plays
an especially important role in sensing and responding to nutrient
status, particularly amino acid availability (16). Downstream
consequences of mTOR activation include effects on transcription,
translation, protein degradation, and cytoskeletal organization (16).
Very little is known regarding the role of mTOR-mediated signaling in
cardiac hypertrophy and cardiac gene regulation. We chose to use amino
acid availability, specifically that of glutamine, as an experimental
tool to probe the role of mTOR signaling in regulating cardiac gene expression.
Glutamine is the most abundant amino acid in the blood, with a
concentration of ~2.5 mM (17, 18). Recent studies
indicate that glutamine is a major gluconeogenic precursor and vehicle for interorgan carbon transport in man (19). It has long been known
that under certain physiological circumstances glutamine serves as
major fuel for the gut (20, 21), the kidney (22), and the immune system
(23-26). Many lines of cultured mammalian cells utilize glutamine, in
preference to glucose, as their major carbon source to meet energetic
and biosynthetic needs (24, 25). Available information suggests that
glutamine serves as a significant source of energy for the heart
(27-30). Glutamine perfusion of the ischemic heart provides
significant protection from damage resulting from loss of energy charge
(31-33). We show here that the addition of glutamine as a major
nutrient for cultured cardiomyocytes produces an increase in myocyte
size and in the organization of actin into myofibrillar arrays. The
maturation of cardiomyocytes is associated with the induction of the
adult isoforms of the contractile proteins, Rat Neonatal Cardiac Myocyte Culture and Glutamine
Treatment--
Neonatal rat cardiac myocytes (1-2 days old) were
isolated as described previously (13) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum (Hyclone). Cells were plated in six-well dishes (21 cm2 per well) at a
density of 5 × 105 cells per well for RNA isolation,
total cellular protein isolation, and transient transfection
experiments (34). For some experiments cells were plated on coverslips
for fluorescent microscopy. After 24 h cells were rinsed and then
maintained for the remainder of the experiment in serum-free DMEM,
supplemented with 1% bovine serum albumin. The cardiac myocytes on the
coverslips were treated by glutamine from 4 to 16 mM in
serum-free DMEM for 24 h. All the cardiac myocytes used for RNA
isolation, total protein isolation, and transient transfections were
treated with 16 mM glutamine. Other cardiac myocytes were
maintained in the serum-free DMEM without glutamine as control. For
some experiments, 1 µM H-89, a protein kinase A
inhibitor, or 5 µM G06983, a protein kinase C inhibitor,
was added to both the control and glutamine-treated cells. Forskolin
was added to both the control and glutamine-treated cells at 1 µM concentration.
Fluorescent Microscopic Techniques--
The morphological
changes in cardiac myocyte size and sarcomere alignment were visually
determined for control and glutamine-treated cells. After 48 h on
coverslips the cells were permeabilized with 0.5% Triton X-100, fixed
in 3.7% formaldehyde (Sigma), and stained for actin with BODIPY
FL phallacidin (Molecular Probes, Inc., Eugene, OR). The fluorescent
images were viewed with an Olympus BX60 fluorescent microscope equipped
with dark-field optics and photographed using a SPOT digital camera
(Diagnostics, Sterling Height, MI).
Total Protein Isolation--
Following the pretreatment by
different inhibitors for 30 min and then with glutamine or
angiotensin II (Ang II) for another 30 min, cardiomyocytes were
extracted with lysis buffer (1% Nonidet P-40, 0.5% sodium
deoxycholate, 0.1% SDS in 50 mM NaCl, 20 mM Tris-HCl, pH 7.6) containing 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml of aprotinin, 10 µg/ml leupeptin, 5 mM benzamidine, 1 mM EDTA, 5 mM
N-ethylmaleimide, 50 mM NaF, 1 mM
sodium orthovanadate, 25 mM glycerophosphate, and 100 nM okadaic acid. The cell lysate was centrifuged at 14,000 rpm for 30 min, and the protein concentration in the supernatant was
determined using BCA (Pierce) reagent (34).
Western Blot Analysis to Detect S6K1 and TSC2--
Total
cellular protein (30-50 µg) from control, Ang II-treated, and
glutamine-treated cells, in the presence or absence of rapamycin (100 ng/ml) or LY294002 (20 µM), were boiled with SDS-sample buffer for 5 min and fractionated in 10% SDS denaturing polyacrylamide gels at 100 volts for 1 h. The separated proteins were transferred from the gel onto nitrocellulose membranes at 100 volts for 1 h.
The blot was blocked with 5% milk in TBST solution for 1 h at
room temperature and then incubated with polyclonal rabbit anti-p70S6
kinase (i.e. S6K1) and anti-phosphothreonine 389-p70S6 kinase, anti-tuberin, and anti-phosphothreonine-1462-tuberin (Cell Signaling Technology, Inc., Beverly, MA) at 1:1500 dilution in 1%
milk-TBST overnight at 4 °C, respectively, and followed 5-min washes
in TBST three times. Finally the blot was incubated for 1 h with
goat anti-rabbit IgG at 1:2000 dilution in 1% milk-TBST for
1 h at room temperature and followed with 5-min washes in TBST
three times. The signal was detected by the ECL detection system
(Amersham Biosciences).
RNA Isolation and Northern Hybridization--
Total RNA was
isolated by using a Trizon RNA isolation kit (Invitrogen). Total
cellular RNA (10 µg/lane) was electrophoresed in 1.0% agarose under
denaturing conditions as described previously (35), and transferred to
nylon membranes. RNA was cross-linked to the membrane by ultraviolet
irradiation and incubated with an Adss1 probes as described
before (13). Following multiple stringent washings the membranes were
subjected to autoradiography for 24-48 h at Preparation of Plasmids and Transient Transfection--
The
reporter construct, 1.9Adss1/CAT, has been described before
(13). The putative cAMP response element-binding protein binding site
within 1.9Adss1/CAT was disrupted using a PCR site-directed mutagenesis kit from Stratagene (13). The consensus CRE, TGACGTCA, was
mutated to TGtgGTCA as published previously (35). Rat neonatal cardiac
myocytes were cultured as above, transfected with plasmid vectors, and
assayed for CAT activity and Quantitative Reverse Transcriptase-PCR Analysis--
Specific
quantitative assays were developed for rat Statistic Analysis--
All values are expressed as mean ± S.E. Data were analyzed for statistical significance using GraphPad
Prism software. Statistical significance was determined by Student's
t test or analysis of variance test. A value of
p < 0.05 was interpreted to mean that observed
experimental differences were statistically significant.
Glutamine Induces Physiological Cell Growth and Maturation--
To
study the role of glutamine in the growth and differentiation of
cardiac myocytes, cells were incubated in serum free minimal medium in
the presence of different concentrations of glutamine (4-16
mM). After 24 h the cells were fixed and subsequently
stained with the immunofluorescent probe, BODIPY phallacidin, to
identify actin filaments. Dramatic differences between the control and glutamine-treated cardiac myocytes were evident, not only in size but
also in the organization of actin from punctate cores of actin characteristic of immature cardiac myocytes into striated arrays of
well organized mature actin filaments. (Fig.
1). These alterations were
accompanied by a significant increase in protein content (75.4 ± 4.8 to 113.67 µg ± 3.3/106 cells,
p < 0.05) and RNA content (1.85 ± 0.85 to 3.32 µg ± 0.55/106 cell, p < 0.05) in
the glutamine-treated cells. These results indicate that the addition
of glutamine to minimum essential medium leads to an increase in
cardiomyocyte size (hypertrophy) and the organization of actin into
myofibrillar arrays (maturation). Similar results were obtained with
certain other amino acids, including alanine, aspartate, asparagine,
and glutamate (data not shown). Because glutamine is the most abundant
amino acid in the circulation and because of the special importance of
glutamine in energy metabolism and nitrogen metabolism we chose to
focus on glutamine for the remainder of our studies.
To evaluate the impact of glutamine on the pattern of cardiomyocyte
gene expression we used quantitative PCR to determine the abundance of
specific mRNAs encoding a diverse group of proteins associated with
cardiac hypertrophy. We initially examined mRNAs encoding various
cardiac contractile proteins, a metabolic enzyme, a peptide hormone,
and a transcription factor. The results (Fig. 2) show that the abundance of cardiac
Glutamine Stimulates Increased Transcriptional Activity of the
Adss1 Gene--
ADSS1 is an enzyme of purine nucleotide synthesis that
functions at a metabolic branch point controlling the synthesis of adenine nucleotides. Glutamine is a required biosynthetic precursor and
intermediate in adenine nucleotide synthesis. Additionally, Adss1 gene expression in cardiomyocytes is stimulated in
response to cardiac hypertrophic stimuli (13, 40). For these reasons we
chose Adss1 as a marker gene to study the cardiac
transcriptional response to glutamine. We initially determined the
effect of glutamine on the abundance of Adss1 mRNA in
cultured rat neonatal cardiac myocytes. Northern hybridization showed
that the abundance of Adss1 mRNA was elevated relative
to control cardiomyocytes by 6 h of glutamine exposure and
continued to increase for at least 24 h (Fig.
3A). To determine whether the
increased abundance of Adss1 mRNA reflected increased
transcription of the Adss1 gene we conducted transfection
experiments with Adss1/CAT reporter constructs. Transfection
assays showed that Adss1 promoter activity increased
6-7-fold in response to glutamine (Fig. 3B). These results imply that intracellular signaling pathways link the presence of
glutamine with transcriptional activation of the Adss1
gene.
Activation of the Adss1 Promoter by Glutamine Is Mediated through
the Protein Kinase A Pathway--
Previous studies indicate that
glutamine controls gene expression through cyclic AMP-mediated
signaling pathways in epithelial cells (21). In an effort to identify
signaling pathways associated with the glutamine induction of
Adss1 gene expression in cardiomyocytes, we tested the
effects of forskolin (an activator of adenylyl cyclase) and glutamine
on Adss1 promoter activity. In addition, we tested the
effect of protein kinase A and protein kinase C inhibitors on
Adss1 promoter activity in the presence and absence of
glutamine. Transfection assays showed that forskolin treatment alone
increased Adss1 transcriptional activity ~4-fold.
Glutamine alone resulted in a 6-fold induction. Glutamine and forskolin
together increased Adss1 promoter activity in an additive
way for a total of 11-fold. The protein kinase C inhibitor (G06983) had
no effect on Adss1 promoter activity, suggesting that
protein kinase C is not involved in the activation of the
Adss1 promoter by glutamine (data not shown). In contrast,
the protein kinase A inhibitor, H-89, completely blocked the activation
of the Adss1 promoter by glutamine but had no effect on its
basal expression in the absence of glutamine (Fig.
4). These results suggest that the
protein kinase A signaling pathway contributes to the induction of
Adss1 promoter activity by glutamine. However, although H-89
is well known as a PKA inhibitor, it is also a potent inhibitor of
p70S6K1, a growth-regulated kinase under the control of mTOR. Thus, it
is possible that the mTOR signaling pathway is involved in the
activation of Adss1 gene expression by glutamine.
Activation of the Adss1 Promoter by Glutamine Is Mediated in Part
by the mTOR Signaling Pathway--
Studies in yeast (41) and
Xenopus laevis (42) have highlighted the
importance of mTOR in the cellular response to glutamine availability.
Recent studies have linked the mTOR pathway with cAMP-induced
transcriptional events suggesting an interplay between mTOR and cAMP
nutrient signaling pathways (43-45). To explore the role of mTOR in
the regulation of Adss1 gene expression we determined the
effect of rapamycin, an inhibitor of mTOR activity, on the induction of
Adss1 promoter activity following glutamine treatment. Transfection assays showed that glutamine treatment resulted in a
4-fold induction of Adss1 promoter activity and that this
induction was inhibited by ~50% by the presence of rapamycin (Fig.
5). Similar results were obtained with
angiotensin II in the absence and presence of rapamycin (Fig. 5). These
results suggest that mTOR activation contributes to the induction of
Adss1 gene expression in response to glutamine, a nutrient,
and angiotensin II, a growth factor.
A key downstream mediator of mTOR activation is the protein kinase,
p70S6K1 (46-48). Activation of p70S6K1 is characterized by the
phosphorylation of a specific threonine residue at amino acid position
389. The phosphorylation of p70S6K1 at threonine 389 is a feature
commonly used to monitor the activation status of mTOR (48). We, too,
used this approach to determine the effect of glutamine treatment on
mTOR activity. Treatment of serum-starved cardiomyocytes with
angiotensin II or glutamine resulted in the phosphorylation of p70S6K1
at Thr-389 (Fig. 6A). In each
case the increased phosphorylation of p70S6K1 was blocked by the
presence of rapamycin (Fig. 6A). These results provide
confirmation that the mTOR signaling pathway is activated in response
to treatment with Ang II or glutamine. The PI3 kinase inhibitor,
LY294002, blocked the phosphorylation of p70S6K1 at Thr-389 induced by
Ang II but not by glutamine. These results indicate that Ang II
activates mTOR through PI3 kinase signaling pathway, whereas glutamine
does not.
To further assess the role of mTOR signaling in regulating
Adss1 gene expression we assessed the phosphorylation status
of tuberin, an upstream regulator of mTOR activity (14). Tuberin serves
as a negative regulator of mTOR activity, a feature that is prevented
when tuberin is phosphorylated at Thr-1462 (49). Thus activation of
mTOR is expected to be associated with the phosphorylation of tuberin.
The impact of Ang II or glutamine treatment on the phosphorylation of
tuberin was determined using a phosphopeptide-specific antibody that
specifically recognized phosphorylation at Thr-1462. The results
presented in Fig. 6B show enhanced phosphorylation of
tuberin in response to Ang II or glutamine treatment. These findings
provide additional evidence that Ang II or glutamine treatment of
serum-starved neonatal cardiomyocytes results in the activation of the
mTOR signaling pathway.
A CRE in the 5' Flanking Region of the Adss1 Gene Is Essential for
Induction by Glutamine--
The research presented above indicates
that glutamine activates Adss1 gene expression
through signaling pathways involving increased intracellular
cyclic AMP and the activation of protein kinases, PKA, mTOR, and
p70S6K1. PKA and p70S6K1 act upstream of transcription factors that
activate gene expression through the use of CREs associated with target
genes (44, 45). Consistent with the potential involvement of these
pathways we identified a CRE in the promoter region of the
Adss1 gene. To determine whether this site is critical for
the glutamine-mediated transcriptional induction of the
Adss1 gene an Adss1 reporter construct with a mutationally destroyed CRE site was prepared. Transfection assays showed that site-directed mutation of CRE in the Adss1
promoter region completely abolished the activation of the
Adss1 promoter by glutamine (Fig.
7). Mutation of the CRE site did not
lower basal expression of the Adss1 reporter construct.
These results indicate that the CRE associated with the
Adss1 promoter region is required for activation by
glutamine.
Considerable attention has been devoted to the study of glucose
and fatty acids as metabolic fuels for the heart (50-52). Very little
attention has been given to the role of amino acids as cardiac fuels
and their impact on cardiac energy metabolism and gene expression. Here
we show that glutamine, the most abundant amino acid in blood, can
induce cardiomyocyte growth and maturation accompanied with increased
abundance of mRNAs encoding the adult isoforms of contractile
proteins ( For much of the research reported here we used the Adss1
gene as a reporter to monitor the cardiomyocyte transcriptional
response to the presence of glutamine. ADSS1 functions at a critical
branch point in purine nucleotide metabolism where it controls the
synthesis of adenine nucleotides (AMP, ADP, and ATP) from the purine
nucleotide intermediate IMP (53). Glutamine is an essential source of
nitrogen in two reactions leading to the synthesis of adenine
nucleotides, providing two of the four nitrogens making up the purine
ring. Because of the importance of ADSS1 in cardiac adenine nucleotide metabolism and the essential role of glutamine in adenine nucleotide synthesis we chose the Adss1 gene for more in depth analysis
of the effects of glutamine on cardiac gene expression. The
Adss1 gene is well suited for our studies, because
considerable information is available regarding transcription factors
regulating Adss1 gene expression in the heart (13, 36, 53).
The Adss1 gene is activated early in the development of the
cardiac lineage and is massively up-regulated during the neonatal
period to achieve very high levels of expression in the postnatal heart
(36, 53). Developmental control of Adss1 gene expression is
achieved through the use of a cardiac-specific enhancer that confers
proper developmental activation and neonatal enhancement in the cardiac
lineage (36, 53). The cardiac enhancer region contains essential
binding sites for numerous well known cardiac transcription factors
including NKX2.5, GATA4, MEF2C, E12, HAND1, and HAND2 (36, 53).
Adss1 gene expression is also induced by a variety of
hypertrophic stimuli including electrical stimulation (13), angiotensin
II (40), and aortic banding (40). In these cases Adss1 gene
activation is mediated through the action of NFAT transcription factors
acting downstream of calcineurin and calmodulin. Adss1
reporter constructs have proven to be sensitive and reliable markers of
cardiac gene expression in cardiomyocyte transfection and transgenic
mouse experiments (36). Our results suggest that the Adss1
gene should serve as an excellent reporter to assess the mechanisms by
which PKA and mTOR signaling pathways regulate the cardiac
transcriptional response to glutamine.
mTOR is an evolutionarily conserved nutrient sensor that directs the
cellular response to nutrient status (15, 16, 48), especially the
availability of amino acids. Recent studies have also identified
tuberin (the product of the TSC2 gene) as a nutrient sensor
(14, 49). In a complex with hamartin (product of the TSC1
gene), tuberin functions as a negative regulator of mTOR, a role that
is prevented by phosphorylation of tuberin at position Thr-1462 (14).
The mechanism by which tuberin and mTOR sense nutrient availability
remains a mystery (14). Our identification of PKA and mTOR kinases as
upstream regulators of Adss1 gene expression in response to
glutamine provides a clue regarding the mechanism by which mTOR and
tuberin function as nutrient sensors. We have shown here that the
presence of glutamine, as the major nutrient, activates the cAMP
signaling pathway. Others have shown that the activation of PKA leads
to the activation of Akt/PKB (54, 55), a protein kinase that is
regulated by a multitude of mitogenic signals (56). Very recent studies
(49, 57) indicate that Akt/PKB directly phosphorylates tuberin, a
modification that results in the dissociation of tuberin from hamartin,
and relieves the negative control of mTOR mediated by the
tuberin/hamartin complex. On the basis of these considerations we
hypothesize in Fig. 8 that mTOR and
tuberin sense nutrient availability via the cAMP signaling pathway
through which the activation of PKA results in the activation of
Akt/PKB. Akt/PKB, in turn, phosphorylates tuberin leading to the relief
of mTOR suppression by the complex consisting of tuberin and hamartin
(TSC2 and TSC1, respectively). Activation of mTOR results in the
activation of a variety of signaling pathways (e.g.
phosphorylation of p70S6K1 at threonine 389), leading to cell growth.
In the context of this hypothesis it is noteworthy that other nutrients
such as glucose (58, 59) and glucosamine also activate the cAMP
signaling pathway. Thus, our hypothesis provides a very general
molecular and metabolic mechanism by which mTOR and tuberin sense
nutrient status and regulate the cellular response to this
information.
-myosin heavy chain and
cardiac
-actin, and the metabolic enzymes, muscle carnitine
palmitoyl transferase I and muscle adenylosuccinate synthetase (ADSS1). Adss1 gene expression was induced ~5-fold in
glutamine-treated rat neonatal cardiac myocytes. The induction was
mediated through the protein kinase A and mammalian target of
rapamycin signaling pathways and required a cyclic AMP response element
associated with the promoter region of the Adss1 gene.
These results highlight glutamine as a major nutrient regulator of
cardiac gene expression and identify protein kinase A and mammalian
target of rapamycin signaling pathways as mediators of the
cardiomyocyte transcriptional response.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-myosin heavy chain
(
-MHC)1 and cardiac
-actin. The adult isoforms of the contractile proteins provide
greater contractility making it possible to pump more blood per
contraction than the fetal isoforms (4, 5). These features make the
physiological hypertrophic heart more efficient and powerful.
Physiological hypertrophy also occurs during the neonatal period as the
heart adapts to the increased energy demands of postnatal life (6) and
during pregnancy in response to increased maternal cardiac demand (7).
Prolonged hypertrophy, usually secondary to other pathology
(e.g. hypertension), often leads to irreversible
cardiomyopathy and heart failure. The pathological hypertrophic
response is characterized by the transcriptional activation of fetal
genes encoding contractile proteins such as
-MHC and skeletal
-actin (8, 9). The synthesis of atrial natriuretic factor (ANF) is
also induced, presumably mimicking the cardiac response to pressure
overload (10). These changes are believed to reflect a change in gene
expression intended to achieve an "energy sparing" rather
than a "contractile efficiency" status and illustrate the fact that
cardiac gene expression is closely linked to energy demand (11). The
intracellular signaling pathways that mediate the cardiac hypertrophic
response are poorly understood as are the molecular mechanisms
controlling whether a hypertrophic response is pathological or physiological.
-MHC and cardiac
-actin, features associated with physiological hypertrophy. Markers
of the pathological hypertrophic response, such as
-MHC, skeletal
-actin, and ANF, are not induced. However, the adult isoforms of two metabolic enzymes, muscle carnitine palmitoyl transferase I
(CPT-1) and muscle adenylosuccinate synthetase (ADSS1), were also
induced. These are the muscle-specific isoforms of enzymes associated with cardiac fatty acid metabolism and adenine
nucleotide metabolism, respectively. Adss1 gene expression
is induced ~5-fold in glutamine-treated rat neonatal cardiac
myocytes. The induction is mediated through the protein kinase A (PKA)
and mTOR signaling pathways and requires a cyclic AMP response element
(CRE) associated with the promoter region of the Adss1 gene.
These results highlight glutamine as a major nutrient regulator of
cardiac gene expression and show that PKA and mTOR signaling pathways
are required for the cardiomyocyte transcriptional response.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C.
-galactosidase as described (13,
36).
-MHC,
-MHC, skeletal
-actin, cardiac
-actin, c-fos, ANF, muscle
CPT-1, and cyclophilin, respectively, from published sequences
(37-39). In each case 100 ng of total RNA (extracted as described
above) was reverse transcribed for 30 min at 42 °C with 400 nm
specific reverse primer, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer, pH 8.3 (at
20 °C), 500 µM deoxynucleotides and 0.1 mU reverse
transcriptase (Superscript II; Invitrogen) in a total volume of 20 µl. Subsequently, 8 µl of the reverse transcriptase reaction was
used for quantitative two-step PCR (95 °C for 1 min, followed by 40 cycles of 95 °C for 12 s, 60 °C for 1 min) in the presence
of reverse primer, 100 nM specific fluorogenic probe, 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris buffer, pH 8.3 (at 20 °C), 200 µM
deoxynucleotides, and 1.2 units of Taq polymerase (Sigma) in
a final volume of 50 µl.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Glutamine induces growth and maturation of
rat neonatal cardiac myocytes. Rat neonatal cardiomyocytes
were cultured in the serum-free medium without and with glutamine
(4-16 mM). After 24 h, cardiomyocytes were fixed and
stained with BODIPY FL phallacidin to visualize actin. The original
photos were taken under ×400 magnification. Bars, 500 µm.
-actin and
-MHC mRNAs increased in response to the presence
of glutamine. Messenger RNA encoding CPT-1, a mitochondrial transport
protein associated with fatty acid metabolism, was also induced by the
presence of glutamine. The abundance of mRNAs encoding skeletal
-actin and
-MHC, fetal isoforms characteristic of hypertrophic
cardiomyopathy, did not change significantly following glutamine
treatment. Evaluation of mRNA from control and glutamine-treated
cells also showed that the cardiac hypertrophic marker gene encoding
the polypeptide hormone, ANF, was not induced. The abundance of c-fos
mRNA increased slightly in response to the presence of glutamine.
Overall, these results show that stimulation of cardiomyocyte growth
and maturation by glutamine was accompanied by increased expression of
genes encoding the adult isoforms of cardiac proteins (e.g.
cardiac
-actin,
-MHC, and muscle CPT-1), with no effect on the
fetal isoforms. Genes normally induced as a result of pathological
hypertrophy, i.e. those encoding skeletal
-actin,
-MHC, and ANF, were not induced in response to the presence of
glutamine as a major nutrient.
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Fig. 2.
Glutamine as a nutrient regulator of mRNA
abundance in rat neonatal cardiac myocytes. Total RNA was isolated
from rat neonatal cardiomyocytes without ( ) and with (+) glutamine.
Quantitative PCR was performed to determine the mRNA concentrations
of
-HMC (A),
-MHC (B), cardiac
-actin
(C), skeletal
-actin (D), c-fos
(E), ANF (F), and M-CPT-1 (G). The
results are expressed as the mean ± S.E. of four independent
determinations. *, p < 0.05.
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Fig. 3.
Glutamine-stimulated increased
Adss1 mRNA abundance and increased transcription
of the Adss1 gene. A, total RNA was
isolated from rat neonatal cardiomyocytes treated without and with
glutamine for up to 48 h. Adss1 mRNA abundance was
determined by Northern blot hybridization using an Adss1
cDNA probe. The results are representative of three independent
experiments. B, 1.9Adss1/CAT was co-transfected
with -galactosidase plasmid into rat neonatal cardiomyocytes without
(
) and with (+) glutamine. The expression of 1.9Adss/CAT
construct was expressed as a -fold induction over that without
glutamine (
). The results are expressed as mean ± S.E. of three
independent determinations. *, p < 0.05.
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Fig. 4.
Transactivation of the Adss1
promoter by glutamine is through the protein kinase A
pathway. 1.9Adss1/CAT was co-transfected with a
-galactosidase construct into cardiomyocytes without (
) and with
(+) glutamine treatment. Cultures were maintained in the absence or
presence of Forskolin (Fsk) or H-89 (protein kinase A
inhibitor). The expression of the 1.9Adss1/CAT construct was
expressed as a -fold induction over that observed from cultures lacking
the glutamine treatment. The results are expressed as mean ± S.E.
of three independent determinations. *, p < 0.05 versus control without glutamine treatment and **,
p < 0.05 versus glutamine treatment.
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Fig. 5.
Activation of the Adss1
promoter by glutamine or Ang II is mediated by mTOR
signaling. 1.9Adss1/CAT was co-transfected with a
-galactosidase construct into cardiomyocytes without (
) and with
(+) glutamine or Ang II treatment. Cultures were maintained in the
absence or presence of 100 ng/ml of rapamycin, an inhibitor of mTOR.
The expression of the 1.9Adss1/CAT construct in
glutamine-treated cells was expressed as a -fold induction over that
seen with cells lacking glutamine treatment. The results are expressed
as mean ± S.E. of three independent determinations. *,
p < 0.05 versus control without glutamine
treatment and **, p < 0.05 versus glutamine
or Ang II treatment.
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Fig. 6.
Phosphorylation of S6K1 and tuberin following
glutamine or Ang II treatment. Cardiac myocytes were serum-starved
overnight, pretreated with rapamycin (100 ng/ml) or LY294002 (20 µM) for 30 min, and then treated with glutamine or Ang II
for 30 min. Cellular extracts were analyzed by Western blotting
analysis using phosphopeptide-specific and phosphorylation-independent
antibodies to S6K1 or tuberin as described under "Materials and
Methods." A, Western blot analysis showing phosphorylation
of S6K1 at Thr-389 in the presence and absence of glutamine or Ang II.
The effect of rapamycin or LY294002 on phosphorylation is also shown.
B, Western blot analysis showing phosphorylation of tuberin
specifically at Thr-1462 in control cells and cells treated with
glutamine or Ang II. Data shown are from one of two sets of similar
results.
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Fig. 7.
A cyclic AMP response element in the 5'
flanking region of the Adss1 gene is essential for induction by
glutamine. Either a wild-type 1.9Adss1/CAT construct or
one with a mutant CRE was co-transfected with a -galactosidase
construct into the cardiomyocytes in the presence or absence of
glutamine treatment. The transcriptional activity of the wild-type and
CRE mutant 1.9Adss1/CAT constructs was expressed as -fold
inductions over that obtained from cells lacking glutamine treatment.
The mutation of CRE site in Adss1 promoter significantly
inhibits the activation of reporter gene in response to glutamine. The
results are expressed as mean ± S.E. of three independent
determinations. *, p < 0.05 versus control
without glutamine treatment and **, p < 0.05 versus glutamine treatment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-MHC and cardiac
-actin) and metabolic enzymes (CPT-1
and ADSS1). The Adss1 gene was chosen for further analysis,
because considerable information is available regarding the regulation
of this gene in the heart (36, 53). Our results revealed the importance
of the protein kinase A and mTOR signaling pathways in the
glutamine-mediated induction of Adss1 gene expression. A
cyclic AMP response element located in the promoter region is required
for glutamine-induced Adss1 gene activation and likely
functions downstream of PKA and mTOR signaling pathways. The results
reported here identify glutamine as a major nutrient regulator of
cardiac gene expression and show that PKA and mTOR signaling
pathways are associated with the cardiomyocyte transcriptional response.
View larger version (29K):
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Fig. 8.
Signaling pathways mediating the cardiac
transcriptional response to glutamine and Ang II. We have shown
here that mTOR signaling is a common feature of both glutamine-induced
and Ang II-induced Adss1 gene activation in neonatal
cardiomyocytes. According to our hypothesis, glutamine activates mTOR
via PKA signaling, whereas Ang II activates mTOR through PI3 kinase
signaling. Glutamine- and Ang II-induced signaling converge on Akt/PKB,
an upstream regulator of mTOR. The Adss1 transcriptional
response to PKA and mTOR signaling relies on a cyclic AMP response
element associated with the gene. In previous studies we have shown
that Ang II-mediated activation of Adss1 gene expression
requires calcineurin/NFAT signaling (13). The Adss1
transcriptional response to Ang II requires an NFAT binding site. The
results presented here highlight the fact that the cardiac
transcriptional response to nutrient and growth factor signaling relies
on the mTOR signaling pathway. See text for more details.
CREM, CRE-binding protein modulator; PDK, PI3
kinase-dependent kinase.
Numerous calcium-sensitive signaling pathways are activated in cardiac
myocytes in response to hypertrophic stimuli (12). Forced activation of
calcium-sensitive signaling pathways is sufficient to induce myocyte
hypertrophy in vitro and in vivo (12). Thus, it
is reasonable to assume that calcium signaling plays a key role in
controlling cardiac gene expression in many forms of hypertrophy. However, it is also clear that several calcium-independent pathways (e.g. Gq, RAS, PI3 kinase, p38) mediate key aspects
of the hypertrophic response (60). We show here that mTOR signaling is
a common feature of both glutamine-induced physiological hypertrophy
and Ang II-induced pathological hypertrophy (Fig. 8). The common use of
mTOR signaling by glutamine and Ang II presumably reflects the central
role of mTOR signaling in controlling cell growth in response to
nutrients and growth factors. We have shown here that glutamine
activates PKA, which, in turn, can activate mTOR signaling through
Akt/PKB (Fig. 8). That is, the mTOR pathway senses nutrients via PKA
acting on Akt/PKB, an upstream regulator of mTOR (Fig. 8). The
mechanism by which PKA senses nutrient signaling is unknown but may
involve the AMP-activated kinase. Using Adss1 transcription
reporter constructs we have shown that a cyclic AMP response element is
required for the cardiac transcription response to glutamine.
Additional downstream requirements for mTOR-mediated transcriptional
regulation have not been identified. Ang II can also activate mTOR
signaling but appears to do so through the PI3 kinase pathway (Fig. 8).
In addition to the mTOR pathway, Ang II can also activate calcium
signaling, which mediates the cardiac transcriptional response through
NFAT binding sites associated with hypertrophic target genes (12). In
this regard, we have shown previously that Ang II induction of
Adss1 gene expression is sensitive to inhibition by
cyclosporin A (an inhibitor of calcineurin) and requires an intact NFAT
binding site in the promoter region of the Adss1 gene. The
results presented here highlight the fact that, like glutamine, Ang II
also activates the mTOR signaling pathway in mediating the Ang II
hypertrophic response. Our results indicate that the Adss1
gene will serve as an excellent reporter to identify components of the
mTOR signaling pathways required for the cardiac transcriptional
response to the hypertrophic effects of nutrients and growth factors.
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ACKNOWLEDGEMENT |
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We are grateful to Dr. Stanislaw Stepkowski for supplying us with rapamycin.
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FOOTNOTES |
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* This work was supported in part by grants from the American Heart Association (to R. E. K.) and from the National Institutes of Health (to H. T.).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.
§ Supported by National Institutes of Health Training Grant T32-HD07324. To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, 6431 Fannin, Houston, TX 77030. Tel.: 713-500-5039; Fax: 713-500-0652; E-mail: Yang.Xia@uth.tmc.edu.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M208500200
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ABBREVIATIONS |
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The abbreviations used are: MHC, myosin heavy chain; CPT-1, carnitine palmitoyltransferase 1; ANF, atrial natriuretic factor; CRE, cyclic AMP response element; mTOR, mammal target of rapamycin; NFAT, nuclear factor of activated T cell; TSC, tuberous sclerosis complex; PKA, protein kinase A; PI3 kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; Ang II, angiotensin II.
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