© 1997 American Heart Association, Inc.
Articles |
Involvement of Transcriptional and Posttranscriptional Mechanisms in Cardiac Overload–Induced Increase of B-Type Natriuretic Peptide Gene Expression
From the Department of Pharmacology and Toxicology (J.M., H.T., M.M., H.R.) and Physiology (O.V.), Biocenter Oulu, University of Oulu (Finland).
Correspondence to Heikki Ruskoaho, MD, Department of Pharmacology and Toxicology, University of Oulu, Kajaanintie 52 D, FIN-90220 Oulu, Finland. E-mail heikki.ruskoaho{at}oulu.fi
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Abstract The induction of atrial and ventricular B-type natriuretic peptide (BNP) gene expression is one of the earliest events occurring during hemodynamic overload. To examine the molecular mechanisms for increased BNP gene expression during cardiac overload, we studied the induction of the BNP gene expression compared with that of atrial natriuretic peptide (ANP) in a modified perfused rat heart preparation. An increase in right atrial pressure of 5 mm Hg resulted in a 1.4-fold (P<.05) and 2.2-fold (P<.01) increase in BNP mRNA levels after 1 and 2 hours, respectively, whereas ANP mRNA levels remained unchanged. Stretching for up to 2 hours also significantly increased right atrial immunoreactive BNP (ir-BNP) levels (from 15.8±2.2 to 20.1±1.2 ng/mg, P<.05). Actinomycin D (10 µg/mL), a transcriptional inhibitor, completely inhibited the stretch-induced increase in atrial BNP mRNA levels at 1 hour (P<.05) and 2 hours (P<.001), whereas a protein synthesis inhibitor, cycloheximide (90 µg/mL), had no effect on basal or direct mechanical stretch-induced increase in right atrial BNP mRNA levels. Furthermore, we examined the role of tyrosine kinase and protein kinase C activities in acute mechanical stretch–induced increase in BNP synthesis. Tyrosine kinase inhibitors lavendustin A (1 µmol/L) and tyrphostin A25 (3 µmol/L) and protein kinase C inhibitors staurosporine (30 nmol/L) and chelerythrine (1 µmol/L) prevented the stretch-induced increase in right atrial ir-BNP concentrations at 2 hours. In addition, chelerythrine inhibited the increase of right atrial BNP mRNA levels stimulated by cardiac overload. These results demonstrate that the early increase of BNP mRNA levels by mechanical stretch results from increased transcriptional activation and is independent of protein synthesis. Our results also suggest that protein kinase C and tyrosine kinases activities may be involved in coupling cardiac overload to alterations in atrial BNP synthesis.
Key Words: cardiac gene expression • mechanical load • natriuretic peptide • transcription
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Introduction |
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Atrial natriuretic peptide, BNP, and CNP are the known members of the mammalian natriuretic peptide system. Whereas ANP is predominantly expressed in the atria of the normal adult heart, BNP is produced and released into the peripheral circulation from the atria and ventricles.1,2 Raised plasma levels of ANP and BNP are observed in conditions associated with cardiac pressure and volume overload, such as chronic heart failure, acute myocardial infarction, and essential hypertension, as well as in chronic renal failure.3-7 Although the plasma concentration of BNP is
1/6 the plasma concentration of ANP in healthy men, BNP levels are
increased to a greater degree than are ANP levels in patients with
congestive heart failure and even surpass ANP levels in severe
cases.3,4 Ventricular levels of BNP mRNA are
substantially increased in response to chronic cardiac overload in the
human heart.8,9 Similarly, transcription of the BNP gene
and BNP peptide levels in the ventricles are increased in experimental
models of cardiac overload, including spontaneously hypertensive
rats2,10-14 and rats with myocardial infarction produced by
coronary ligation.15 Furthermore, we have recently
demonstrated that atrial and ventricular BNP mRNA and
peptide levels, in contrast to those of ANP, increase rapidly in
response to acute hemodynamic overload in normotensive
and hypertensive rats.11 These observations of gene
expression and release suggest that BNP may have an important role in
the maintenance of cardiovascular homeostasis;
thus, it is of great importance to characterize the mechanisms
regulating BNP gene expression in response to acute and chronic
increase in cardiac workload. The 5'-flanking promoter region of the ANP gene has been intensively studied,16,17 and the AP-1 binding site is shown to be a critical cis element in the induction of the ANP gene expression in myocyte hypertrophy.18 An analysis of the 5'-flanking sequences of the BNP gene has also indicated the presence of consensus sequences for some well-characterized transcriptional proteins, including several GATA motifs and AP-1–like elements.19,20 Deletion of the distal GATA motifs has been shown to reduce rat BNP promoter activity 4-fold, and deletion of the AP-1–like motif decreased promoter activity another 4-fold.19 Unlike ANP, however, the 3'-untranslated region of BNP mRNA contains several AUUUA sequences21,22 that may be involved in the translation-dependent rapid mRNA degradation.23 Thus, the elevation of BNP mRNA in response stimuli may occur at the transcriptional level as well as at the posttranscriptional level. Indeed, observations that the treatment of neonatal rat ventricular cardiocytes with cycloheximide increases BNP mRNA levels24,25 and enhances the elevation of BNP mRNA levels stimulated by TPA24 suggest a role for posttranscriptional regulation and involvement of a regulatory protein(s). On the other hand, in the cell culture models of myocyte hypertrophy, treatment with actinomycin D, a transcriptional inhibitor, reduces the stimulatory effect of phorbol esters24 and endothelin-125 on BNP mRNA levels. To date, however, there are no reports of the molecular mechanisms responsible for mechanical load–induced activation of the BNP gene expression.
In the present study, we examined the involvement of transcriptional and posttranscriptional regulation of the BNP gene compared with that of the ANP gene in response to mechanical loading of right atria by using the previously described isolated perfused rat heart preparation.26 Since mechanical loading has been reported to activate several signaling pathways, including the activity of PKC and PTK in cultured cardiac myocytes,27 we also examined their involvement in stretch-induced changes in atrial BNP gene expression. Furthermore, we studied the effect of right atrial stretch on c-fos mRNA levels.
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Materials
The chemicals and supplies used in the present study were as follows: formaldehyde and guanidine isothiocyanate (Fluka Chemie AG); CsCl (Serva Feinchemica GmbH & Co); agarose NA (Pharmacia); heparin (Leiras); synthetic rat ANP99-126, actinomycin D, cycloheximide, and staurosporine (Sigma Chemical Co); BAS 85 nitrocellulose membrane (Schleicher & Schuell); [32P]dCTP, radioiodine, and rat [125I]ANP (Amersham); x-ray films (Eastman Kodak); lavendustin A and chelerythrine (Research Biochemicals Inc); and tyrphostin A25 (Calbiochem-Novabiochem Intl). Other chemicals were from Sigma.
Experimental Animals
Male 2-month-old Sprague-Dawley rats (260 to 330 g) were
from the Center for Experimental Animals at the University of Oulu. The
rats were housed in plastic cages in a room with a controlled 40%
humidity and temperature of 22°C, and a 6-h/18-h (on/off)
environmental light cycle was maintained. The experimental design was
approved by the animal experimentation committee of the University of
Oulu.
Isolated Perfused Heart Preparation
The isolated perfused rat heart preparation was similar to that
described previously.26,28 The aorta was cannulated above
the aortic valve, and retrograde perfusion was begun with a modified
Krebs-Henseleit bicarbonate buffer, pH 7.4, equilibrated with
O2/CO2 (95:5) at 37°C. Final concentrations
of the salts in the buffer were as follows (mmol/L): NaCl 113.8,
NaHCO3 22.0, KCl 4.7, KH2PO4 1.2,
MgSO4 · 7H2O 1.1, CaCl2
· 2H2O 2.5, and glucose 11.0. Variations in the perfusion
pressure, arising from changes in coronary vascular resistance,
were recorded on a Grass polygraph (model 7DA, Grass Instruments)
with a pressure transducer (model MP-15, Micron Instruments) situated
on a side arm of the aortic cannula. Isometric force of contraction was
recorded by a strain-gauge transducer (model FTO3, Grass
Instruments) connected to the Grass polygraph. The heart rate was
counted from contractions by means of a Grass tachograph. The hearts
were put under resting tension of 2.0 g and stimulated (11 V, 0.5
milliseconds) with a Grass stimulator (model S88, Grass Instruments) to
increase the heart rate to a level of 300 bpm. The hearts were paced
via the pulmonary artery cannula and the cannula in the
inferior vena cava. During the equilibration period (40
minutes), the hearts were perfused with a peristaltic pump (Minipuls 3,
model 312, Gilson) at a constant flow rate of 5 mL/min. Drugs were
infused via an infusion pump (Secan PSA 55, Skyelectronics S.A.). The
intracardial concentrations of lavendustin A, tyrphostin A25,
chelerythrine, and staurosporine were 1, 3, 1, and 0.03
µmol/L, respectively.
Right atrial pressure was recorded on a Grass polygraph via a cannula (PE-60) in the inferior vena cava connected to a pressure transducer (model MP-15). After a 10-minute control period, right atria were stretched for 1 or 2 hours by elevating the level of the pulmonary artery cannula tip. Right atrial pressure was kept constant during the experiments at the desired level by means of fine adjustment of the level of the pulmonary artery cannula tip.28 Drugs were infused continuously throughout the stretch period. Immediately after perfusion, the right auricles from control and stretched hearts were carefully removed, weighed, immersed in liquid nitrogen, and stored at -70°C until assayed.
Isolation and Analysis of Cytoplasmic RNA
RNA was isolated from right atria by the guanidine
thiocyanate–CsCl method.29 For the RNA Northern blot and
dot-blot analysis, 3.0-µg samples of the RNA from the right
atria were transferred to the Schleicher & Schuell BAS 85
nitrocellulose membrane. A 390-bp fragment of rat BNP cDNA
probe2 (a generous gift from Dr Kazuwa Nakao, Kyoto
[Japan] University School of Medicine), a full-length rat ANP cDNA
probe30 (a generous gift from Dr Peter L. Davies, Queen's
University, Kingston, Canada), a full-length cDNA probe complementary
to GAPDH,31 an oligonucleotide probe
complementary to rat 18S ribosomal RNA,32 and a cDNA probe
for rat c-fos made by reverse-transcriptase polymerase chain
reaction were labeled with [32P]dCTP with a Quick Prime
kit (Pharmacia LKB Biotechnology). The membranes were hybridized
overnight at +42°C in 5x SSC (1x SSC contains 0.15 mol/L
NaCl and 0.015 mol/L trisodium citrate, pH 7), 0.5% SDS, 5x
Denhardt's solution, 50% formamide, and 100 µg/mL sheared
herring sperm DNA. After hybridization, the membrane was washed in
0.1x SSC and 0.1% SDS three times for 20 minutes at +50°C and
exposed at -70°C to x-ray film with Cronex Lighting Plus
intensifying screens (DuPont). Autoradiograms generated
by dot blots were scanned with a densitometer (Millipore Corp Imaging
Systems). The hybridization signal of ANP mRNA and BNP mRNA was
normalized to that of GAPDH mRNA or 18S mRNA for each sample to correct
for potential differences in loading and/or transfer.
Radioimmunoassay of BNP and ANP
For the BNP radioimmunoassay, the atrial guanidine thiocyanate
extracts were diluted 100-fold and assayed without extraction. For the
ANP radioimmunoassay, the atrial guanidine thiocyanate extracts were
diluted 5x104-fold. The tissue extracts were incubated in
duplicates of 100 µL with 100 µL of the specific rabbit
BNP11 or ANP antiserum.33 Synthetic rat
BNP51-95 (BNP-45) and synthetic rat ANP99-126,
ranging from 0 to 500 pg/tube, were incubated as standards. The
tracer was prepared by chloramine-T iodination of synthetic rat
[Tyr0]BNP51-95 or ANP99-126,
followed by reverse-phase HPLC purification. After incubation for 48
hours at +4°C, the immunocomplexes were precipitated with sheep
antiserum directed against rabbit gamma globulin in the presence of 500
µL of 8% polyethylene glycol 6000, pH 7, followed by
centrifugation at 3000g for 30 minutes. The
sensitivities of the BNP and ANP assays were 2 fmol/tube and 1
fmol/tube, respectively. Fifty percent displacement of the
respective standard curve were at 16 and 25 fmol/tube. The
intra-assay and interassay variations for both assays were <10% and
15%, respectively. Serial dilutions of tissue extracts showed
parallelism with the standards. The ANP antiserum recognized ANP and
proANP with equal avidity but did not cross-react with BNP or CNP
(<.01%). The BNP antiserum did not recognize ANP or CNP (<0.1%).
The reagents for BNP radioimmunoassay were generously supplied by Dr
Kazuwa Nakao (Kyoto [Japan] University School of Medicine).
Statistical Analysis
The results are expressed as mean±SEM. Student's t
test was used for the comparison between two groups. Differences at the
95% level were considered statistically significant.
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Results |
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Effect of Mechanical Loading on Tissue BNP mRNA and ir-BNP Levels
We first determined in the isolated perfused rat heart preparation the degree of mechanical stretch that would mimic the effect of acute pressure load on BNP gene expression observed previously in vivo,11 in which vasopressin and phenylephrine infusions in conscious spontaneously hypertensive rats produced a 1.5- to 3.6-fold increase in atrial BNP mRNA levels and values peaked at 2 hours. Therefore, the level of the pulmonary artery cannula tip was raised to increase right atrial pressure 4, 5, or 7 mm Hg above the baseline level for 2 hours. Stretching of the atria for 2 hours at a right atrial pressure level that was 4 mm Hg above the baseline level resulted in a 2-fold increase in the BNP mRNA levels (P<.01), whereas right atrial ir-BNP concentration remained unchanged (Fig 1
). When the right atrial
pressure was increased 5 mm Hg above the baseline level for 2
hours, significant increases in both right atrial BNP mRNA (2-fold,
P<.01) and ir-BNP (22%, P<.05) levels were
observed. Stretching of the right atria for 2 hours at a pressure level
7 mm Hg above the baseline level resulted in a 2.1-fold
(P<.001) increase in BNP mRNA levels, whereas right atrial
ir-BNP concentration did not increase significantly (Fig 1). Mechanical
stretch of the right atria had no effect on tissue ANP mRNA and ir-ANP
levels (Fig 1). On the basis of these findings, a right atrial pressure
level of 5 mm Hg was chosen for further experiments, since this
pressure level increased both atrial BNP mRNA and ir-BNP levels as did
acute pressure overload induced by vasopressin and
phenylephrine in conscious rats.11
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Transcriptional Induction of BNP by Mechanical Loading
To examine the effect of inhibiting transcription on basal and
stretch-stimulated BNP mRNA levels, isolated perfused rat hearts were
treated with actinomycin D (10 µg/mL) for 1 hour and 2 hours.
Previously, concentrations of 5 and 10 µg/mL of actinomycin D
have been reported to inhibit the early induction of BNP gene
expression stimulated by endothelin-1 and phenylephrine in
cultured rat ventricular myocytes.25,34 The
baseline hemodynamic parameters (heart
rate, contractile force, and perfusion pressure) did not differ between
the groups, but infusion of actinomycin D resulted in an increased
perfusion pressure in the end of the experiments (Table 1). This increase in the perfusion
pressure (11 mm Hg), however, was small compared with the change
in perfusion pressure (90 mm Hg) needed to induce protein
synthesis in the perfused rat heart.35 The infusion of
actinomycin D alone reduced the basal BNP mRNA level by 54%
(P<.05) at 2 hours (Figs 2 and 3). When actinomycin D was infused
during mechanical loading of the right atria, it completely inhibited
the stretch-induced activation of BNP gene at 1 hour
(P<.05) and 2 hours (P<.001). Administration of
actinomycin D also reduced the basal c-fos mRNA levels and
completely inhibited the stretch-induced activation of c-fos
gene at 2 hours (Fig 3), whereas it had no significant effect on right
atrial ANP mRNA levels (Figs 2 and 3).
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Effect of Protein Synthesis Inhibition on Early Induction of BNP
Gene Expression
To examine the effect of protein synthesis inhibition on the
mechanical stretch–induced BNP gene activation, cycloheximide was
infused during perfusion for 1 or 2 hours. The dose of cycloheximide
(90 µg/mL) was designed to ensure rapid and effective
inhibition. This concentration was 16-fold higher than that used by
Gordon et al36 to inhibit synthesis of contractile proteins
in perfused rat heart preparations. The baseline
hemodynamic variables (heart rate, contractile
force, and perfusion pressure) did not differ between the groups and
were stable for the period of time that was used in these experiments
(Table 1
). Infusion of cycloheximide without stretch had no effect on
BNP mRNA levels (Figs 2 and 3). Furthermore, cycloheximide treatment
during right atrial stretch had no statistically significant effect on
BNP mRNA levels compared with vehicle infusion. Nor did cycloheximide
infusion have any significant effect on the right atrial ANP mRNA
levels (Fig 2). In contrast, administration of cycloheximide for 2
hours increased the basal c-fos mRNA levels and resulted in
an increased activation of c-fos gene in response to direct
mechanical stretch (Fig 3).
Effect of Lavendustin A and Staurosporine on
Stretch-Induced Increase of BNP Synthesis
To examine the role of tyrosine kinase and PKC activities in the
mechanical stretch–induced activation of the BNP gene, vehicle, PTK
inhibitor lavendustin A (1 µmol/L), or PKC
inhibitor staurosporine (30 nmol/L) was
infused. The doses of protein kinase inhibitors were chosen
in order to avoid marked effects on basal natriuretic
peptide synthesis and hemodynamic variables. The
inhibition constant of staurosporine for PKC is 0.7
nmol/L, and it is 2- to 10-fold greater for other kinases,
whereas lavendustin A selectively inhibits PTK at low micromolar
concentrations.37 To validate the dose of
staurosporine as a PKC inhibitor compound, we
have previously determined under these experimental conditions the
concentration of staurosporine necessary to block the
coronary vasoconstrictor and ANP secretory effects induced by
46 nmol/L of the phorbol ester TPA, a concentration known to
stimulate PKC activity in the isolated perfused rat heart
preparation.38 In those experiments,
staurosporine at concentrations from 10 to 100
nmol/L completely abolished the increase in perfusion pressure
and the ANP release produced by phorbol ester.12,39
Staurosporine also inhibited the ANP secretory and cardiac
effects of the PKC-activating peptide endothelin-1.39 To
exclude the possibility that lavendustin A inhibits PKC, we studied the
effects of lavendustin A and phorbol ester TPA both alone and together
on ANP secretion in the isolated perfused rat heart preparation. At a
concentration of 1 µmol/L, lavendustin A failed to affect
phorbol ester–induced ANP secretion, whereas at considerable higher
concentrations (26 µmol/L), it decreased basal ANP
secretion (data not shown). The atria were stretched, and protein
kinase inhibitors were infused for 1 or 2 hours. Perfusion
pressure, heart rate, right atrial pressure, and
contractility were measured continuously throughout the
experiments. The baseline hemodynamic
parameters did not differ between vehicle- and drug-infused
groups (Table 2).
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As shown in Fig 4, atrial stretch
produced a 2.1-fold increase in right atrial BNP mRNA levels at 2
hours accompanied by an increase (40%, P<.05) in the
atrial ir-BNP concentration. Mechanical stretch for 2 hours had no
effect on right atrial ANP mRNA or ir-ANP levels (Fig 4). Infusion of
lavendustin A, staurosporine, or their combination for 2
hours had no significant effect on the basal or stretch-stimulated BNP
mRNA levels but inhibited completely the stretch-induced increase in
right atrial ir-BNP concentrations (Fig 4). Infusion of
staurosporine alone (without stretch) had no effect on the
right atrial ir-BNP levels, whereas lavendustin A infusion for 1 hour
produced a 1.6-fold (P<.05, data not shown) increase in the
atrial ir-BNP concentration. Furthermore, a combined infusion of
lavendustin A and staurosporine for 1 hour increased the
right atrial ir-BNP concentration by 30% (P<.05, data not
shown). Administration of lavendustin A, staurosporine, or
both in experiments without stretch or during stretch had no effect on
right atrial ANP mRNA or ir-ANP levels (Fig 4).
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Effect of Tyrphostin A25 and Chelerythrine on Stretch-Induced
Increase of BNP Synthesis
To examine further the role of PTK and PKC activities in the
mechanical stretch–induced BNP gene activation, vehicle, PTK
inhibitor tyrphostin A25 (3 µmol/L), or PKC
inhibitor chelerythrine (1 µmol/L) was
infused. Tyrphostin A25 is a cell-permeable competitive
inhibitor of substrate binding on PTK, and the inhibition
constant of tyrphostin A25 for the epidermal growth factor receptor
tyrosine kinase is 3 µmol/L.40 Chelerythrine
is a potent and specific inhibitor of PKC, with
half-maximal inhibition of the kinase occurring at 0.7
µmol/L.41 The atria were stretched, and tyrphostin
A25 and chelerythrine were infused for 2 hours, as described above for
lavendustin A and staurosporine experiments. Perfusion
pressure, heart rate, right atrial pressure, and
contractility were measured continuously throughout the
experiments. The baseline hemodynamic
parameters did not differ between vehicle- and drug-infused
groups (Table 3).
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Stretching of the right atria for 2 hours resulted in increases in both
BNP mRNA (P<.001) and ir-BNP (P<.05) levels of
the right atria. Infusion of tyrphostin A25 for 2 hours had no
significant effect on the basal or stretch-stimulated increase of
atrial BNP mRNA levels, but it decreased the stretch-induced increase
in right atrial ir-BNP concentrations (Fig 5). Infusion of chelerythrine inhibited
completely the stretch-induced increase of both right atrial BNP mRNA
and ir-BNP levels. Infusion of tyrphostin A25 slightly decreased right
atrial ir-ANP levels, but the administration of tyrphostin A25 or
chelerythrine in experiments without stretch or during stretch had no
effect on atrial ANP mRNA or ir-ANP levels (Fig 5).
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In the present study, we examined the regulation of BNP compared with ANP gene expression by using an in vitro model of cardiac overload in which atrial cells are acutely stimulated by mechanical loading, which is the predominant stimulus for BNP gene expression and secretion in vivo. During the distension of the right atria, the rapid induction of the BNP gene occurred in a manner characteristic of an immediate-early gene, whereas ANP mRNA and peptide levels remained unchanged. The mechanical load–induced rapid activation of the BNP gene appeared to be the result of transcriptional upregulation and did not require de novo synthesis of trans-acting regulatory factors. In addition, the blockade of the mechanical stretch–stimulated increase in immunoreactive BNP concentration by PKC and PTK inhibitors suggests that these protein kinases may be involved in coupling cardiac overload to alterations in BNP peptide synthesis. Furthermore, since ANP mRNA levels did not change with the increased cardiac overload, the ANP gene expression is less sensitive, or the regulation of the ANP gene expression may be different from that of the BNP in response to acute cardiac overload.
Recent studies have demonstrated the differential expressions of several cardiac-specific genes during increased cardiac overload and myocyte hypertrophy.17,42-44 The early genetic response (within 2 hours) to mechanical loading in cardiac myocytes includes transcription of immediate-early response genes, such as c-fos, c-myc, c-jun, and EGR-1. Upregulation or subtype switching of the contractile protein gene expression and augmented expression of the ANP gene occur later during ventricular hypertrophy.17,42 Recently, we have demonstrated that in vivo pressure overload induced by vasopressin and phenylephrine infusions rapidly stimulates BNP gene expression in the hearts of normal and hypertensive rats within 1 hour,11 thus mimicking the rapid induction of proto-oncogenes in response to hemodynamic overload.43,44 The present study succeeded in reproducing the distinct characteristics of the BNP and ANP gene expression and allowed us to specifically examine the regulatory mechanisms of the BNP gene expression during cardiac overload. The time course of the BNP gene expression during mechanical loading observed in the present study compares favorably with that reported in previous in vitro studies using cultured neonatal rat ventricular cardiocytes24,25,34 but differs from that reported by Bruneau and de Bold,45 in which BNP mRNA levels in isolated rat atrial preparations remained near control levels throughout the 4-hour mechanical stretch period.
The elevation of BNP gene expression in response to mechanical loading could result from transcriptional and posttranscriptional mechanisms or from the combination of both mechanisms. The observations that treatment of neonatal rat ventricular cardiocytes with phenylephrine or endothelin-1 enhances BNP transcript stability and that cycloheximide, a protein synthesis inhibitor, increases BNP mRNA levels suggest a role for posttranscriptional regulation.24,25,34 On the other hand, the treatment with endothelin-125 and phenylephrine34 could evoke a rapid increase in the BNP mRNA level (within 1 to 3 hours) in cultured rat ventricular myocytes even in the presence of cycloheximide, suggesting that the increase in BNP mRNA levels is not due to posttranscriptional events. In agreement with these studies, treatment of neonatal rat ventricular cardiocytes with actinomycin D, a transcriptional inhibitor, reduces the stimulatory effect of phorbol ester24 and endothelin-125 on BNP mRNA levels. Thus, the elevation of the BNP mRNA levels studied in cell culture models of myocyte hypertrophy appears to be mediated by both transcriptional and posttranscriptional events.
To test whether the mechanical stretch–stimulated early induction of BNP gene expression is dependent on protein synthesis, we infused cycloheximide during the right atrial distension. Cycloheximide treatment without stretch had no effect on BNP mRNA levels, and stretch-induced activation of the BNP gene was not diminished during cycloheximide treatment. This finding that stretch-induced rapid augmentation of BNP mRNA levels is independent of protein synthesis is a hallmark of immediate-early genes.43,44 To further evaluate the molecular mechanisms of BNP gene expression, actinomycin D was infused during stretch. We found that the basal levels of BNP mRNA decreased by 54% at 2 hours and that the stretch-stimulated activation of BNP gene at 1 hour and 2 hours was completely inhibited by actinomycin D treatment. Taken together, these results indicate that the early induction of BNP mRNA by mechanical stretch results from the enhanced transcription and is independent of protein synthesis. Also degradation of BNP mRNA during mechanical stretch appears to be translation independent, whereas a translation-dependent destabilization mechanism may be involved in the cell culture models of myocyte hypertrophy treated with activators of PKC.24,25,34
The signaling mechanisms by which hemodynamic overload induces a rapid increase of BNP mRNA and peptide levels have not yet been elucidated. In rat neonatal ventricular myocytes, it has been shown that the expression of the BNP gene increases as an effect of activators of PKC, such as TPA,24,25,46 phenylephrine,25,34,47 or endothelin-1.25 Levels of BNP transcripts also increase in cultured rat atrial cells46 and in isolated rat atria45 stimulated by endothelin-1. Moreover, endothelin-1,46,48 phenylephrine,45 and phorbol ester24,46,48,49 stimulate the secretion of ir-BNP in cultured cardiocytes, suggesting that PKC activation may be a proximal signaling pathway in the regulation of BNP synthesis. In cultured cardiomyocytes, mechanical stretch activates, in addition to PKC, a phosphorylation cascade, including tyrosine kinases and mitogen-activated protein kinases.27,50,51 In the present study, we showed that the PTK inhibitors (lavendustin A and tyrphostin A25) as well as PKC inhibitors (chelerythrine and staurosporine) completely prevented the mechanical stretch–stimulated increase in atrial ir-BNP levels, suggesting that tyrosine kinase and PKC activities may be involved in the transduction pathway between the mechanical load and the acute increase of tissue BNP peptide levels. The mechanisms by which PTK and PKC inhibitors influence the load-induced increase of atrial BNP peptide levels remain to be studied but might be explained mainly by decreased translational efficiency or capacity rather than changes in transcription, since only the PKC inhibitor chelerythrine statistically significantly decreased the stretch-induced increase of tissue BNP mRNA levels. A further explanation for the decreased tissue BNP levels in the presence of PTK and PKC inhibitors could be that mechanical load increases the release of BNP from the atria. However, in contrast to this hypothesis, protein kinase inhibitors did not significantly alter or even decreased mechanical load–induced BNP peptide release into the perfusate (data not shown). The lack of effect of staurosporine on atrial BNP mRNA levels compared with the attenuation of tissue BNP gene expression in the presence of chelerythrine infusion may be related to the moderate low dose of staurosporine that we used in the present study in order to avoid suppressing the activities of other protein kinases. Staurosporine has been found to inhibit the activity of a variety of protein kinases, such as cAMP- and cGMP-dependent kinases and myosin light chain kinase, at high doses.37
The expression of c-fos has been found to be rapidly induced by mechanical stress both in vivo and in vitro.43,44 As expected, in the present study mechanical loading of the right atria resulted in increased c-fos mRNA levels. In agreement with the classical definition of an immediate-early gene, the stretch-induced increase of c-fos gene expression was inhibited by infusion of actinomycin D. Furthermore, infusion of cycloheximide resulted in increased levels of c-fos mRNA but not BNP mRNA levels compared with the infusion of vehicle. It has been shown previously that stretching of the isolated rat right atria increases c-fos mRNA levels but leaves BNP mRNA levels unchanged45 and that cycloheximide causes a strong induction of c-fos mRNA levels in cultured rat ventricular cardiocytes.34 Thus, although both c-fos and BNP mRNA levels are activated by mechanical loading, the results of the present and previous studies suggest that mechanisms of the activation of BNP gene expression by mechanical stretch appear to be different from those of c-fos. In addition, since cycloheximide treatment markedly enhanced c-fos gene expression but BNP mRNA levels increased similarly in vehicle- and cycloheximide-treated hearts, our results indirectly suggest that the activation of c-fos gene expression may not be involved in the activation of BNP gene expression after mechanical stretch under these experimental conditions.
In conclusion, these results indicate that the gene expression of BNP takes place with many of the characteristics of an immediate-early gene. We have shown that the mechanical stretch–induced early increase of BNP mRNA levels results from increased transcription and is independent of protein synthesis. Our results in isolated perfused rat heart preparations have also demonstrated for the first time that PTK and PKC inhibitors disrupt mechanical load–induced atrial BNP peptide synthesis, suggesting that activities of PKC and PTK may represent important components of the cardiac mechanotransduction pathway.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by the Medical Research Council of the Academy of Finland, Sigrid Juselius Foundation, Emil Aaltonen Foundation, and Ida Montin Foundation. We thank Tuula Lumijärvi, Sirpa Rutanen, Tuula Räisänen, and Marja-Leena Vainikka for expert technical assistance.
Received April 17, 1997; accepted August 26, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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