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-myosin heavy
chain promoter activity
Division of Endocrinology, Department of Medicine, North Shore-Long Island Jewish Research Institute, New York University School of Medicine, Manhasset, New York 11030
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Contractile activity of the cardiac
myocyte is required for maintaining cell mass and phenotype, including
expression of the cardiac-specific 
-myosin heavy chain (-MHC)
gene. An E-box hemodynamic response element (HME) located at position
47 within the -MHC promoter is both necessary and sufficient to
confer contractile responsiveness to the gene and has been shown to
bind upstream stimulatory factor-1 (USF1). When studied in
spontaneously contracting cardiac myocytes, there is enhanced binding
of USF1 to the HME compared with quiescent cells, which correlates with
a threefold increase in -MHC promoter activity. A molecular
mechanism by which contractile function modulates -MHC
transcriptional activity may involve signaling via phosphorylation of
USF1. The present studies showed that purified rat USF1 was
phosphorylated in vitro by protein kinase C (PKC) and cAMP-dependent
protein kinase (PKA) but not casein kinase II. Phosphorylated USF1 by
either PKC or PKA had increased DNA binding activity to the HME.
PKC-mediated phosphorylation also leads to the formation of USF1
multimers as assessed by gel shift assay. Analysis of in vivo
phosphorylated nuclear proteins from cultured ventricular myocytes
showed that USF1 was phosphorylated, and resolution by two-dimensional
gel electrophoresis identified at least two distinct phosphorylated USF1 molecules. These results suggest that endogenous kinases can
covalently modify USF1 and provide a potential molecular mechanism by
which the contractile stimulus mediates changes in myocyte gene transcription.
ventricular myocytes; contractile activity; protein kinase C; cAMP-dependent protein kinase; E-box
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EXPRESSION OF
THE cardiac-specific -myosin heavy chain (-MHC) gene is
regulated by multiple humoral and hemodynamic factors (10, 21,
32). These include the hemodynamic workload imposed on the heart
and, specifically, the contractile activity of the ventricular myocyte
(12, 23, 25). Our previous studies (24, 38)
have identified a hemodynamic responsive element (HME) at position 47
of the -MHC promoter that contains an E-box motif (CACGTG) that
binds upstream stimulatory factor-1 (USF1). Overexpression of cloned
rat USF1 increased -MHC promoter activity in contracting ventricular
myocytes but failed to stimulate the promoter in contractile-arrested cardiomyocytes, suggesting that posttranslational modification of USF1
may be required for its enhanced transcriptional activity (38).
USF1 and the related polypeptide USF2 (43 and 44 kDa, respectively)
belong to the basic helix-loop-helix leucine zipper family of
transcription factors. These nuclear transacting factors are involved
in the expression of several tissue-specific and developmentally regulated genes, including cardiac myosin light chain-2
(22), -MHC (38), ribosomal RNA
(8), and cyclin B1 (5). The USFs form
homodimers, heterodimers, and multimers and have been shown to interact
with the basal transcription factor complex TFIID (13,
37). Numerous studies (17, 29) have shown that phosphorylation of transcription factors is an effective mechanism by
which DNA binding and transcriptional activity can be modulated. Cardiomyocyte nuclear factors MEF2C and GATA4, believed to mediate hypertrophic-induced gene transcription, have been shown to be phosphorylated by casein kinase II and extracellular signal-regulated kinase (ERK), respectively, resulting in altered DNA binding and transcriptional activities (15, 16, 20). Similarly, one study (3) showed how the transcriptional activity of USF1
could be modified by phosphorylation. A recent report (7)
showing phosphorylation of USF1 by the stress responsive p38 kinase may have significance in the cardiomyocyte response to stress-induced hypertrophic gene transcription.
Protein phosphorylation has been shown to either enhance DNA binding activity and transcriptional efficacy as described for the muscle-specific MEF2C (20) or to disrupt DNA binding as shown for Max, c-Myc, and c-Myb (2, 26, 29). Phosphorylation of amino acid residues within the prebasic and basic regions of the DNA binding domain of the transcription factors disrupts DNA binding (2), whereas phosphorylation within the leucine zipper region results in stabilization of the multimeric complexes and enhances DNA binding and transactivation (17, 34). Rat USF1 contains three protein kinase C (PKC) consensus phosphorylation sites within the leucine zipper domain, which may enhance its DNA binding potential, as well as two sites within the prebasic region that may act to disrupt DNA binding.
Thus the present study was undertaken to determine whether the transcriptional activity of USF1 in cardiomyocytes could be regulated by phosphorylation. In vitro studies with bacterially expressed USF1 showed that exogenous PKC and cAMP-dependent protein kinase (PKA), but not casein kinase II, phosphorylated USF1, enhanced its DNA binding affinity, and promoted multimerization. Phosphoprotein analysis of nuclear extracts from cultured cardiomyocytes showed that USF1 was phosphorylated in vivo, suggesting a potential mechanism of transcriptional regulation.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Construction of pGEX-rUSF1 expression vector. The pGEX expression system (Pharmacia; Piscataway, NJ) was used to synthesize the glutathione S-transferase (GST)-rUSF1 fusion protein. Full-length rat (r) USF1 cDNA (1.6 kb) that we had previously cloned (38) was ligated in frame into the BamH I-Xho I site of pGEX-4T-3 and verified by DNA sequence analysis. The plasmid (pGEX-rUSF1) was transformed into BL21 Escherichia coli for fusion protein expression according to the manufacturer's instructions (36).
Expression and purification of rUSF1 in bacteria.
BL 21 E. coli harboring the pGEX-rUSF1 plasmid were grown to
A600 
1 and then induced with 0.75 mM
isopropyl-
-thiogalactoside at 25°C for an additional 6 h.
Cells were collected by centrifugation, resuspended in ice-cold
phosphate-buffered saline (50 µl/ml initial culture), and then
disrupted by sonication. Triton X-100 was added to 1% final
concentration and gently mixed for 30 min. The supernatant containing
the GST-rUSF1 fusion protein was recovered by centrifugation at 12,000 g for 10 min at 4°C and then incubated with glutathione Sepharose 4B (50:1), as described by the manufacturer (Pharmacia). The
GST-rUSF1 fusion protein was eluted from the Sepharose beads by
incubation with glutathione. Alternatively, the Sepharose-bound GST-rUSF1 fusion protein was first digested with thrombin protease to
dissociate rUSF1 from GST, and then the purified rUSF1 was eluted from
the beads. The eluted proteins were concentrated by ~10-fold using
microconcentrators (3 kDa MWCO, Amicon; Beverly, MA), and the protein
concentration was determined by Lowry assay.
Western blot analysis. Nuclear extracts were prepared as previously described (24, 38). HeLa cell nuclear extracts (10 µg), GST-rUSF1 fusion protein (2 µg), and thrombin protease-liberated rUSF1 (2 µg) were resolved by electrophoresis on 1% sodium dodecyl sulfate (SDS)-10% polyacrylamide gels. The proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Bio-Rad; Hercules, CA) at 250 mA for 2 h, then incubated overnight at 4°C in 3% milk-10 mM Tris · HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20 (TBST). Rabbit anti-human USF1 antibody (Santa Cruz Biotechnology; Santa Cruz, CA) diluted 1:2,000 in 3% milk-TBST was incubated with the membrane at room temperature for 2 h and then washed extensively before incubation with the secondary antibody, goat anti-rabbit IgG conjugated to horseradish peroxidase (1:5,000 dilution). The signal was developed with the use of a chemiluminescence reagent (Renaissance, DuPont; Boston, MA) and detected by exposure to X-ray film. The same membrane was stripped and reprobed with anti-GST antibody (1:2,000 dilution) and developed with secondary antibody as described above.
In vitro phosphorylation of rUSF1.
For in vitro phosphorylation reactions, 5 µg of bacterially expressed
rUSF1 protein was added to a 12-µl reaction containing PKC (isoforms
, , and 
) purified from rat brain (0.24 U) (Upstate Biotechnology; Lake Placid, NY) in buffer containing 20 mM
Tris · HCl (pH 7.5), 10 mM MgCl2, 0.5 mM
CaCl2, 100 µg/ml phosphatidyl serine, 20 µg/ml phorbol
12-myristate 13-acetate (PMA), and 10 µM [-32P]ATP
(3,000 Ci/mmol). Control reactions did not contain exogenous protein
kinase. Reaction conditions for PKA (60 U) (Promega; Madison, WI)
contained 40 mM Tris · HCl (pH 7.4), 20 mM Mg-acetate, and 10 µM [-32P]ATP. Reactions were resolved by SDS-PAGE.
-32P]ATP and used for electrophoretic
mobility shift assay (EMSA), as we have previously published
(24).
EMSA.
A deoxyoligonucleotide double-stranded DNA probe (dsHME) used in the
EMSA included the -MHC promoter sequences from 59 to 39
[5'-CAGCAGGCACGTGGAATGAGC-3'], with the rUSF1 binding
sequence underlined (24). The sense DNA strand was 5'
end-labeled with T4 polynucleotide kinase and
[-32P]ATP to ~2 × 106 dpm/pmol,
purified over Sephadex G-25 spin columns, and then annealed to 50-fold
excess of the unlabeled antisense strand. Bacterially expressed and
purified rUSF1 (2 µg) was phosphorylated with either PKA or PKC as
described above except that radiolabeled ATP was not used. Parallel
reactions were carried out in the absence of the kinases, and equal
aliquots of all reactions were compared for DNA binding activity by
EMSA using conditions as previously described (24, 38). To
measure DNA binding activity of the bacterially expressed rUSF1 in the
presence of mammalian cell extract, 2 µg of purified rUSF1 was
incubated with either untreated or heat-inactivated (95°C for 10 min)
rabbit reticulocyte lysate (1.5 µl) (Promega) and analyzed by EMSA.
Cultures of neonatal rat ventricular myocytes and in vivo analysis of phosphorylation of rUSF1. Neonatal rat ventricular myocytes (NRVM) were plated at ~1.5 × 104/cm2 on collagen-coated 60-mm plates and cultured as previously described (24, 25). After 30 h in culture, the medium was replaced by phosphate-free Dulbecco's modified Eagle's medium (GIBCO-BRL; Gaithersburg, MD) for 10 h, followed by the addition of [32P]orthophosphate (9,000 Ci/mmol; 0.1 mCi/ml medium) for 4 h. Cells were washed extensively and harvested, and nuclear extracts were prepared as previously described (24). Extract proteins were desalted and concentrated ~10-fold using microconcentrators (Amicon; Beverly, MA).
In a separate series of experiments, NRVM were transiently cotransfected with control plasmid-galactosidase (pRSVZ) and luciferase reporter plasmids containing either wild-type -MHC promoter sequences (388 to +32) (p388wt) or containing a mutant HME
at position 50 within the context of the 388/+32 bp region of the
promoter (p388
), as we have previously published (24). Minimal promoter constructs containing two copies of either the wild-type or mutant HME element ligated to luciferase (pHMEwt, pHME)
were similarly transiently transfected into NRVM, as previously published (38). After transfection, cells were cultured in
serum-free medium with half of the culture plates treated with PMA
(2 × 10
7 M) for 24 h (or 1 h followed by
23 h culture without PMA) before harvest and analysis of
luciferase and -galactosidase (-gal) activities. Results are
expressed as luciferase luminescence units normalized for -gal
activity in the same volume of cell lysate, as previously described
(24, 38). Some cell cultures were treated simultaneously
with PMA and staurosporine (10 nM), an inhibitor of PKC activity, for
24 h before harvest.
Two-dimensional gel electrophoresis.
Two-dimensional (2D) gel electrophoresis used the mini-Protean II 2-D
system (Bio-Rad; Hercules, CA). Nuclear proteins (10-20 µg) were
solubilized in 2D sample buffer containing 4.75 M urea, 1.0% Triton
X-100, 2.5% -mercaptoethanol, 0.8% 5/8 ampholytes, and 0.2% 3/10
ampholytes (Sigma; St. Louis, MO) and isoelectric focused at 12,000 V-h
in a 4% polyacrylamide tube gel containing 9.5 M urea, 2% Triton
X-100, 1.6% pH 5-8 ampholytes, and 0.4% pH 3-10 ampholytes.
The isofocused proteins were resolved in the second dimension by
electrophoresis in an SDS-10% polyacrylamide slab gel, in which an
additional lane was used to resolve the same nuclear protein sample
only by SDS-PAGE. The resolved 2D and one-dimensional (1D) proteins
were then transferred to PVDF membrane (Bio-Rad). The membrane was
first exposed to phosphorimage analysis (Bio-Rad) and subsequently used
for Western blot analysis with anti-human USF1 antibodies as described
above. To increase the resolution of the phosphorylated isoforms of
USF1, nuclear extracts (10-15 µg) were isofocused on ReadyStrip
IPG pH 4-7 (Bio-Rad) and then subjected to 2D SDS-PAGE
electrophoresis before Western blot analysis, as described above.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Characterization of the bacterially expressed rUSF1.
Rat USF1 was synthesized in bacteria as a GST fusion protein and
purified by glutathione Sepharose affinity chromatography. Figure
1 shows a Western blot of the fusion
protein (GST-rUSF1) as well as the cleaved proteins GST and rUSF1.
Analysis with anti-USF1 antibody (Fig. 1, left) recognized
the fused protein (~64 kDa) and a protein of ~35 kDa in size that
was not recognized by anti-GST antibody (Fig. 1, right),
suggesting that rUSF1 had been appropriately cleaved from GST. The
anti-GST antibody recognized a 29-kDa protein that was not recognized
by the anti-USF1 antibody, indicating that this protein was GST. The
smaller apparent molecular mass of rUSF1 produced in bacteria (~35
kDa) may be the result of altered processing of the protein in bacteria
compared with its expression in eukaryotic cells, as we and others have
previously shown (36, 38).
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In vitro phosphorylation of rUSF1 by PKA and PKC.
To determine whether rUSF1 can be phosphorylated in vitro, the ability
of PKC, PKA, and casein kinase II to phosphorylate the protein was
examined. As shown in the SDS-PAGE in Fig.
2, both PKA and PKC were able to
phosphorylate rUSF1 observed as the ~35 kDa band. Autophosphorylation
of PKA occurred as evidenced by the phosphorylated band at ~40 kDa
(Fig. 2, left, lanes 1 and 2), whereas
autophosphorylation of PKC was not observed (Fig. 2, right).
In contrast to PKC and PKA, rUSF1 was not phosphorylated by casein
kinase II (data not shown).
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Phosphorylation enhances rUSF1 DNA binding activity.
EMSA was used to determine whether the DNA binding affinity of rUSF1
was enhanced by phosphorylation. Bacterially produced rUSF1 bound to
the -MHC E-box element (HME) (Fig. 3,
lane 1) and was prevented from DNA binding by the anti-USF1
antibody (lane 4) similar to rUSF1 derived from
cardiomyocyte nuclear extracts, as we have previously published
(24). We tested the hypothesis that the DNA
binding activity of bacterially expressed rUSF1 may be covalently
modified by factors present in a mammalian cell lysate. Therefore, we
compared binding of bacterially expressed rUSF1 to the HME sequence in
gel shift assays incubated either in the presence of native rabbit
reticulocyte lysate or with heat-inactivated lysate. In Fig. 3, DNA
binding of rUSF1 incubated with native rabbit reticulocyte lysate
(lane 2, +RL) was greater than when incubated with
heat-inactivated lysate (lane 3, +RL), suggesting that
the mammalian cell lysate modified rUSF1 in a manner leading to an
alteration in its DNA binding potential.
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Phorbol esters activate -MHC promoter activity via the
hemodynamic E-box element.
To determine whether a PKC signaling pathway may be
involved in mediating the activation of the -MHC promoter
through the E-box HME enhancer, we treated transiently transfected
cultured neonatal rat ventricular myocytes with the phorbol ester PMA, which is known to activate several PKC isoforms, and measured reporter
gene activity. As shown in Fig. 4,
PMA treatment for 24 h (1-h treatment showed similar results)
stimulated -MHC promoter (p388wt) activity by 3.5-fold,
whereas mutation of the HME enhancer (p388) completely obliterated
the PMA affect. To further determine whether PMA activation of the
-MHC promoter was mediated by the HME element, we transiently
transfected minimal promoter constructs containing two copies of
wild-type or mutant HME. As shown in Fig. 4, PMA treatment increased
pHMEwt activity by twofold and had no stimulatory effect when the HME
enhancer was mutated (pHME). Simultaneous treatment of the
PMA-treated cells with staurosporine completely prevented the
PMA-induced increase in wild-type -MHC promoter activity (data not
shown). Together, these data suggest that PKC-mediated signaling may be
involved in the activation of the -MHC promoter by altering the
binding activity of the HME-binding protein, USF1.
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In vivo phosphorylation of rUSF1.
We determined whether phosphorylation of rUSF1 occurred in vivo using
spontaneously contracting neonatal cardiomyocytes cultured in the
presence of [32P]orthophosphate. The nuclear
phosphoproteins were analyzed by two-dimensional gel electrophoresis as
shown in Fig. 5. Figure 5A
shows the phosphorimage analysis of the nuclear phosphoproteins resolved by 2D analysis (predicted isoelectric point and SDS-PAGE) and
1D SDS-PAGE. Figure 5B shows the same PVDF membrane as in Fig. 5A but analyzed by Western blotting with anti-USF1
antibody. In lane 1D of Fig. 5B, one
immunoreactive band at the predicted molecular mass of ~40 kDa was
recognized by the anti-USF1 antibody. In contrast, at least two
distinct immunoreactive USF1 proteins were resolved at the same
molecular mass by 2D analysis as indicated by the arrowhead and the
arrow, and these proteins isofocused at the predicted isoelectric point
of 5.6.
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-MHC gene.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypertrophic growth of the cardiac myocyte in response to
increased hemodynamic load is characterized by activation of
intracellular signaling pathways, by induction of immediate early and
secondary response genes including the -MHC genes, and by an
accumulation of myocyte-specific proteins (1, 12, 23, 25,
27). A role for cell surface integrins and focal adhesions has
been suggested in transmitting the mechanical hypertrophic signal to
intracellular signaling cascades that include PKC isoenzymes, c-Jun
NH2-terminal kinases, and ERKs, which in turn regulate
nuclear transcription factors (4, 6, 9, 19, 31). Despite
recent studies (28, 33) of these intracellular signaling
pathways, understanding the mechanisms by which extracellular signals
result in altered transcription of relevant cardiomyocyte genes such as
the myosin heavy chains remains unclear.
We previously identified that USF1 bound to an E-box HME within the
proximal region of the -MHC promoter that was necessary and
sufficient to confer contractile responsiveness to the promoter in
ventricular myocytes (24, 38). We also showed that
overexpression of rat USF1 in contracting ventricular myocytes
increased the activity of a minimum -MHC promoter construct
containing the E-box HME; however, in contractile-arrested myocyte
cultures, USF1 overexpression did not stimulate the -MHC promoter,
suggesting that USF1 was modified differently under the two conditions
resulting in distinct transcriptional activities (38). We
therefore, hypothesized that myocyte contractile activity signaling
through intracellular kinase cascades alters phosphorylation of nuclear
USF1, thus acting as a potential mechanism by which mechanochemical
stimuli could be transmitted to alterations in transcriptional activity
of cardiac genes.
Our initial observation showing that the DNA binding activity (as
measured by EMSA) of bacterially expressed rat USF1 was enhanced when
incubated in the presence of rabbit reticulocyte lysate but not when
the lysate was heat inactivated suggested that USF1 was covalently
modified. We subsequently showed that phosphorylation of purified USF1
with exogenous classic PKC isoenzymes (mixture of PKC, -, and
- isoforms from rat brain) not only enhanced its binding to the
E-box HME but also promoted the formation of multimers. This is
analogous to the basic leucine zipper protein, cAMP response element
binding protein, in which PKC phosphorylation induces dimer formation,
whereas PKA does not, and that the degree of phosphorylation of cAMP
response element binding protein complexes modulates its
transcriptional activity (17). Three consensus PKC
phosphorylation sites of rat USF1 (serine-257, -262, and -309) are
located within the leucine zipper domain, which has been shown for
other basic leucine zipper proteins to enhance binding to DNA, whereas
three potential phosphoserine residues within the prebasic domain of
USF1 would tend to disrupt DNA binding (26, 34). Two of
these potential PKC sites (serine-257 and -262) are also consensus
sites for PKA phosphorylation and may provide a mechanism by which
diverse signaling pathways can converge on a single DNA element.
Alternatively, the preferential formation of USF multimers by PKC
phosphorylation may provide a mechanism for distinct transcriptional
effects of these signaling pathways (37). We have shown
here that activation of PKC signaling pathways by treatment of cultured
cardiomyocytes with phorbol esters increased the activity of the
-MHC promoter containing the wild-type E-box enhancer element HME
but not when the HME was mutated. Therefore, together these data
support a potential role for distinct PKC signaling pathways in
regulating USF1-mediated -MHC transcription (25).
Electrical pacing and stretch-induced cardiomyocyte hypertrophy have
been shown to activate one of several kinase cascades including p38
kinase, PKC
, and PKC
, ERKs, or c-Jun NH2-terminal
kinases (18, 30, 31, 35).
In the present study, we determined that phosphorylation of USF1
occurred in vivo in neonatal rat ventricular myocytes grown in culture.
Two-dimensional electrophoretic analysis showed that a significant
fraction (~40-50%) of the total nuclear USF1 was phosphorylated, thus providing a mechanism by which a specific kinase
or phosphatase pathway could determine the transcriptional potential of
USF1. We identified two distinct molecular species of phosphorylated
USF1 as well as nonphosphorylated USF1 in both contracting and
contractile-arrested myocyte cultures. When we compared total
phosphorylated to nonphosphorylated USF1 in contracting myocytes to
that in quiescent cultures, we found a higher phosphorylation ratio in
the noncontracting myocytes. Because site-specific phosphorylation rather than total phosphorylation per se determines transcriptional activity, we must conclude that our observation of increased DNA binding activity of PKC-phosphorylated USF1 as well as increased -MHC promoter activity in response to PMA and attributable to the
HME enhancer reflects distinct site-specific phosphorylation of USF1.
Therefore, increased phosphorylation of USF1 in contractile-arrested myocytes may occur at amino acid residues that result in disruption of
DNA binding. Mutagenesis studies of each of the six potential PKC and
PKA phosphorylation sites within USF1 may determine which intracellular
signaling pathways are important in mediating the transcriptional
activity of USF1 on target promoters.
Protein serine-threonine phosphatases have been associated with the
nuclear envelope of cardiomyocytes (11), and the nuclear trafficking of variants of protein-tyrosine phosphatases has been shown
in response to cellular stress (14), suggesting that
dephosphorylation of nuclear proteins as well as their phosphorylation
may play a role in the present model. Alternatively, induction of
intracellular kinase cascades that may ultimately lead to
phosphorylation of nuclear USF1 have been reported in other models of
hypertrophy, namely, stretch-activated and electrically stimulated
cardiomyocyte hypertrophy (6, 9, 30). Thus the signaling
paradigms in these models may be distinct from the spontaneously
contractile cardiomyocytes used in the present study. USF1 must also
function in combination with numerous other transcription factors
necessary for both inducible and tissue-specific expression of the
-MHC gene (10, 21, 32). Therefore, phosphorylation of
USF1 as well as other nuclear factors, including those of the MEF2 and GATA families, may provide a mechanism by which multiple diverse stimuli can modulate transcriptional activities of specific genes that
would determine cardiomyocyte phenotype (1, 28, 33).
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ACKNOWLEDGEMENTS |
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The authors thank Dr. Irwin Klein for invaluable insight and discussion of these studies.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-56804 and HL-03775 (to K. Ojamaa) and an American Heart Association-New York State Fellowship (to Q. Xiao).
Address for reprint requests and other correspondence: K. Ojamaa, Div. of Endocrinology, North Shore-Long Island Jewish Research Institute, 350 Community Dr., Manhasset, NY 11030 (E-mail: kojamaa{at}nshs.edu).
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.
First published March 28, 2002;10.1152/ajpheart.01085.2001
Received 10 December 2001; accepted in final form 19 March 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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