Contractile activity is required for sarcomeric assembly in
phenylephrine-induced cardiac myocyte hypertrophy
Diane M.
Eble1,2,
Ming
Qi1,2,
Stephanie
Waldschmidt1,2,
Pamela A.
Lucchesi1,2,
Kenneth L.
Byron1,2,3, and
Allen M.
Samarel1,2,3
1 The Cardiovascular Institute and the
Departments of 2 Physiology and
3 Medicine, Loyola University
Chicago Stritch School of Medicine, Maywood, Illinois 60153
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ABSTRACT |
Agonist-induced
hypertrophy of cultured neonatal rat ventricular myocytes (NRVM) has
been attributed to biochemical signals generated during receptor
activation. However, NRVM hypertrophy can also be induced by
spontaneous or electrically stimulated contractile activity in the
absence of exogenous neurohormonal stimuli. Using single-cell imaging
of fura 2-loaded myocytes, we found that low-density, noncontracting
NRVM begin to generate intracellular
Ca2+ concentration
([Ca2+]i)
transients and contractile activity within minutes of exposure to the
1-adrenergic agonist
phenylephrine (PE; 50 µM). However, NRVM pretreated with verapamil
and then stimulated with PE failed to elicit
[Ca2+]i
transients and beating. We therefore examined whether PE-induced [Ca2+]i
transients and contractile activity were required to elicit specific
aspects of the hypertrophic phenotype. PE treatment (48-72 h)
increased cell size, total protein content, total protein-to-DNA ratio,
and myosin heavy chain (MHC) isoenzyme content. PE also stimulated
sarcomeric protein assembly and prolonged MHC half-life. However,
blockade of voltage-gated L-type
Ca2+ channels with verapamil,
diltiazem, or nifedipine (10 µM) blocked PE-induced total protein and
MHC accumulation and prevented the time-dependent assembly of
myofibrillar proteins into sarcomeres. Inhibition of actin-myosin
cross-bridge cycling with 2,3-butanedione monoxime (7.5 mM) also
prevented PE-induced total protein and MHC accumulation, indicating
that mechanical activity, rather than
[Ca2+]i
transients per se, was required. In contrast, blockade of
[Ca2+]i
transients and contractile activity did not prevent the PE-induced increase in cell surface area, activation of the mitogen-activated protein kinases ERK1 and ERK2, or upregulation of atrial natriuretic factor gene expression. Thus contractile activity is required to elicit
some but not all aspects of the the hypertrophic phenotype induced by
1-adrenergic receptor
activation.
calcium; verapamil; signal transduction; fura 2; gene expression; cytoskeleton
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INTRODUCTION |
PROLONGED EXPOSURE to
1-adrenergic agonists produces
a variety of hypertrophic alterations in neonatal rat ventricular
myocytes (NRVM), including increased cell size, sarcomeric protein
assembly, and specific changes in gene expression that are typical of
hemodynamic overload in vivo.
1-Adrenergic receptor
activation also acutely activates cell signaling cascades that involve
the heterotrimeric G protein Gq
(18, 30), mitogen-activated protein kinases (MAPK) (12, 26, 41), and
the small GTPases Ras (18, 40) and Rho (30, 42). However, it is unclear
exactly which steps in the signal transduction pathways are necessary
and/or sufficient for the induction of various aspects of the
hypertrophic phenotype. Whereas either Ras or
Gq
activation by the
1-adrenergic agonist phenylephrine (PE) was sufficient for the induction of atrial natriuretic factor (ANF) gene transcription (a molecular marker of the
NRVM hypertrophy), only Ras activation was necessary and sufficient to
elicit the structural reorganization of cytoskeletal proteins into
sarcomeres (18, 40). In another study, Rho, a stimulator of stress
fiber formation in nonmuscle cells (28), was neither necessary nor
sufficient for PE-induced sarcomeric assembly in NRVM (42). In
contrast, inactivation of Rho A protein by ADP ribosylation in
embryonic chick ventricular myocytes caused sarcomeric disruption in
the absence of other exogenous stimuli (43).
In contrast to studies with
1-adrenergic agonists, we and
others have found that mechanical load in the form of either
electrically stimulated or spontaneous contractile activity induces
NRVM growth in the absence of exogenous agonists (14, 21-24, 31).
The hypertrophic response was very similar to that observed in response
to
1-adrenergic receptor
activation, in that the intrinsic mechanical load generated during
excitation-contraction coupling was sufficient to elicit the
transcriptional activation of the "fetal" genes, ANF (10, 24) and
-myosin heavy chain (MHC) (25, 27, 31), as well as to promote the
assembly of newly synthesized contractile proteins into sarcomeres (3,
24, 31, 32, 35). Of particular interest to us was the observation by
Kimura et al. (16) that
1-adrenergic receptors couple
directly to Ca2+ influx via
voltage-gated, L-type Ca2+
channels, resulting in a marked increase in beating frequency. Earlier
observations by Simpson (38) indicated that
1-adrenergic stimulation was
sufficient to stimulate quiescent, low-density NRVM cultures to
contract. These observations suggested that at least some of the
phenotypic alterations produced by
1-adrenergic stimulation were
secondary to mechanical events generated after receptor activation. In
the present study, we have quantitatively analyzed the role of
Ca2+ influx and contractile
activity on specific features of the hypertrophic phenotype induced by
PE exposure. Data are presented to indicate that PE-induced contractile
activity is required to elicit sarcomeric assembly, but not the
activation of the MAPKs ERK1 and ERK2 or upregulation of ANF gene
expression in response to
1-adrenergic receptor
activation.
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METHODS |
Reagents.
PC-1 tissue culture medium was obtained from BioWhittaker
(Walkersville, MD). DMEM was obtained from GIBCO BRL (Grand Island, NY). Medium 199, Ca2+-free and
Mg2+-free
Hanks' balanced salts (modified) (HBSS), acid-soluble calf skin
collagen, and antibiotic/antimycotic solution were obtained from Sigma
Chemical (St. Louis, MO). Permanox chamber slides and slide wells were
obtained from Nunc (Naperville, IL). Tissue culture plates were
obtained from Costar (Cambridge, MA).
[32P]ATP,
[32P]dCTP, and
[35S]methionine were
purchased from Amersham (Arlington Heights, IL). Fura 2-AM and Pluronic
F-127 were obtained from Molecular Probes (Eugene, OR).
4-(2-Aminoethyl)-benzenesulfonyl fluoride was obtained from Boehringer
Mannheim (Basel, Switzerland). Rabbit polyclonal antibodies to ERK1 and
ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad
(Hercules, CA). All other reagents were of the highest grade
commercially available and were obtained from Sigma and Baxter S/P
(McGaw Park, IL).
Ventricular dissociation and cardiac myocyte isolation.
Animals used in these experiments were handled in accordance with
National Institutes of Health "Guide for the Care and Use of
Laboratory Animals" [Department of Health and Human Services Publication No. (NIH) 85-23, Revised 1985]. Ventricular
myocytes were isolated from the hearts of 2-day-old Sprague-Dawley rats by collagenase digestion, as previously described (31). Released cells
were collected by centrifugation, resuspended in PC-1 medium, and
plated at a density of 400 cells/mm2 onto collagen-coated
plastic 35-, 60-, and 100-mm dishes as well as Permanox chamber slides
or slide wells. They were left undisturbed in a 5%
CO2 incubator (37°C) for
14-18 h. Unattached cells were then removed by aspiration, and
cells were maintained in a 4:1 mixture of DMEM-medium 199 containing
antibiotic/antimycotic solution (myocyte growth medium). Medium was
changed daily.
Measurement of intracellular
Ca2+
concentration.
Myocytes plated onto Permanox chamber slides were maintained in growth
medium with daily medium changes for 2-3 days. The cells were then
loaded with the fluorescent Ca2+
indicator fura 2 by incubating with fura 2-AM [2 µM in a
modified Krebs medium (135 mM NaCl, 5.9 mM KCl, 1.5 mM
CaCl2, 1.2 mM
MgCl2, 11.5 mM glucose, and 11.6 mM HEPES, pH 7.3) supplemented with 0.1% BSA and 0.02% Pluronic F-127
detergent] for 2 h at room temperature, followed by a 1- to 3-h
incubation in Krebs medium alone. Fura 2 fluorescence was then measured
in individual cells using a video microscopy system composed of a Nikon
Diaphot inverted epifluorescence microscope, rotating filter wheel, and
an intensified charge- coupled device video camera (Hamamatsu model
XC77/C2400). The slide was mounted in a chamber (Warner Instrument) on
the stage of the microscope and superfused with media at a rate of
5-10 ml/min. A four-way valve mounted adjacent to the chamber
allows rapid switching of solutions from four gravity-fed reservoirs. The field of cells was excited alternately with 340- and 380-nm light,
and the average brightness of eight individual cells was recorded to
disk. Background fluorescence was determined for each cell at the end
of the experiment by quenching the fura 2 fluorescence for 15 min in
the presence of 1 µM ionomycin and 6 mM
MnCl2 in Ca2+-free medium. After background
fluorescence was subtracted, the 340- and 380-nm values were ratioed
and calibrated in terms of intracellular
Ca2+ concentration
([Ca2+]i).
This procedure allows simultaneous measurement of
[Ca2+]i
in eight individual cells with a temporal resolution of ~0.6 s. All
[Ca2+]i
measurements shown are from experiments conducted at 25°C.
Calibration of fura 2 fluorescence in terms of
[Ca2+]i,
as described previously (4), routinely utilized solutions of known Ca2+ concentration to construct a
standard curve. A look-up table was then prepared for analysis of
fluorescence ratios recorded from cells. The
Ca2+ concentration was calculated
using software (MaxChelator, version 6.60) that accounts for binding of
Ca2+ to each constituent of the
solution. In situ calibration of fura 2 fluorescence by determination
of maximum and minimum ratios (13) from within cells yields similar
calibrated values (data not shown).
Cell area measurements.
Cells were grown on Permanox slide wells and maintained in growth
medium with daily medium changes for 2-3 days. During the last 48 h of this period, the cells were treated with medium alone (control) or
growth medium containing verapamil (10 µM), PE (50 µM), or PE plus
verapamil. The cells were then loaded with fura 2 as described above,
except that the concentration of fura 2-AM was 4 µM, and the
treatments were present in both the loading medium and subsequent wash
medium. The area of the fluorescently labeled cells was then determined
by image analysis using Universal Imaging Image 1 software. A binary
mask was created by setting a threshold brightness that distinguished
the fluorescent cells (illuminated with 380-nm light) from the black
background. The area of the mask for each cell was then determined.
When a cell was in contact with one or more adjacent cells, the area of
the mask was divided by the number of cells. The mean cell area was determined for 120 cells in each of the treatment groups. Absolute area
measurements may be slightly underestimated because very thin areas at
the cell periphery may not have been detected above the background
fluorescence.
Myofibrillar structure.
Cells grown on Permanox slide wells were fixed (10 min, room
temperature) with 2% (wt/vol) paraformaldehyde in sodium PBS, washed (15 min) in 1% (wt/vol) glycine in PBS, and
permeabilized (15 min) with 0.5% (vol/vol) Triton X-100 in PBS.
Myocytes were then stained with FITC-conjugated phalloidin to visualize
F-actin filaments and myofibrillar structure (35). The
phalloidin-stained cells were viewed using a Zeiss model LSM 410 scanning laser confocal microscope. Multiple optical sections ~1 µM
thick were taken of each sample to eliminate out-of-focus fluorescence
of the intensely stained myocytes.
Cellular composition.
For the quantitative analysis of total cellular protein and DNA
content, cells grown on 35-mm dishes were washed twice in HBSS, and 0.2 N perchloric acid (1 ml) was added. The precipitated macromolecules
were then quantitatively scraped from the dishes and collected by
centrifugation (10,000 g, 10 min). The
precipitate was redissolved by incubation (60°C, 20 min) in 250 µl of 0.3 N KOH. Aliquots were then used for analysis of total
protein by the Lowry method using crystalline human serum albumin as
standard, and for DNA using 33258 Hoecht dye and salmon sperm DNA as
standard, as previously described (31). Data are the means of duplicate or triplicate wells from each treatment group for each cell isolation and are expressed as micrograms per dish. For quantitative analysis of
-MHC and
-MHC content, cells were washed twice in HBSS and lysed
in 250 µl of sample buffer [62.5 mM
Tris · HCl, pH 6.8, containing 8% (wt/vol) SDS, 5%
(vol/vol) 2-mercaptoethanol, and 10% (wt/vol) glycerol].
The concentrations of
-MHC and
-MHC isoenzymes were assessed by
SDS-PAGE and silver staining (31). MHC band intensity was quantified by
laser densitometry and compared with the band intensity of purified MHC
standards (0-300 ng). The positions of the
-MHC and
-MHC
bands were confirmed by electrophoresis of
-MHC and
-MHC protein
standards obtained from normal and hypothyroid adult rat hearts,
respectively, and by Western blotting with an anti-MHC antibody that
cross-reacts equally with both isoenzymes (data not shown). Results are
the means of duplicate wells from each treatment group for each cell
isolation and are expressed as micrograms per dish.
Pulse-chase biosynthetic labeling experiments.
MHC degradation in control, PE-treated, and verapamil-treated cultures
was assessed in pulse-chase biosynthetic labeling experiments, as
previously described (3, 32). Cells in 35-mm dishes were incubated (24 h, 37°C) in myocyte growth medium supplemented with 8 µCi/ml
[35S]methionine. At
the end of the pulse-labeling period, cells were rapidly rinsed twice
in HBSS and either harvested by addition of 500 µl SDS sample buffer
or chased for 24 h in growth medium supplemented with 2 mM unlabeled
methionine, or methionine-supplemented growth medium containing PE (50 µM), verapamil (10 µM), or their combination. Cell samples were
then separated by SDS-PAGE on 180-mm-long, 0.7-mm-thick, 7-17%
vertical gradient SDS-polyacrylamide gels. In each experiment, a
constant fraction of the total protein of each culture dish was applied
to individual gel lanes. This ensured that for all pulse-chase
experiments, the amount of radioactivity in MHC declined by decay
rather than by simple dilution. After electrophoresis, gels were
autoradiographed with fluorographic enhancement. Dried gels were
exposed to unflashed Kodak XAR-5 film for varying time periods
(2-4 days) at
80°C. Individual MHC bands on the
autoradiographs were scanned three times, and the average area beneath
the MHC peak was computed by autointegration. Linearity of detection of
radioactivity by fluorography was assessed as previously described
(32). The fractional rate of MHC degradation (MHC
Kd, %/h) for
each condition was estimated by the following formula
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where
ln(MHC AU)0 and ln(MHC
AU)24 are the natural logarithms
of the average absorbance [in arbitrary absorbance units
(AU)] of the MHC bands at times 0 and 24 h of the chase. MHC
Kd values were
converted to apparent half-lives
(t1/2; in h)
according to the following formula
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MAPK Western blots.
Myocytes plated onto 100-mm dishes were maintained in myocyte growth
medium for 48 h. Cells were then switched to fresh growth medium or
growth medium containing verapamil (10 µM). After an additional 1 h,
myocytes were stimulated (5 min) with phorbol 12-myristate 13-acetate
(PMA, 200 nM) or PE (50 µM) in the presence or absence of verapamil.
Thereafter, cells were scraped into 0.9 ml of MAPK extraction buffer
[10 mM HEPES, pH 7.4, containing 50 mM sodium pyrophosphate, 50 mM sodium chloride, 5 mM EDTA, 5 mM EGTA, 50 mM sodium fluoride, 0.1 mM
sodium vanadate, 0.01% Triton X-100, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride].
Aliquots of the cell extracts (25 µg of total protein) were separated
by SDS-PAGE and transferred to nitrocellulose membrane by
electroblotting. The blots were probed with a mixture of polyclonal
antibodies directed toward ERK1 (44 kDa) and ERK2 (42 kDa). Primary
antibody binding was detected by horseradish peroxidase-conjugated goat
anti-rabbit secondary antibody using the enhanced chemiluminescence kit
from Amersham.
ANF promoter activity.
An expression plasmid consisting of 3003 bp of upstream regulatory
sequences of the rat ANF gene linked to the gene encoding firefly
luciferase was used to analyze ANF transcription in control, PE-treated, and verapamil-treated myocytes using both the calcium phosphate method and adenoviral-assisted transfection (17). P3003ANF-luc expression plasmid consisted of 3003 bp of upstream regulatory sequences of the rat ANF gene linked to the gene encoding firefly luciferase and was kindly provided by Dr. Andrew Thorburn, University of Utah, and Dr. Kenneth Chien, University of California, San Diego. A constitutively active Rous sarcoma virus long terminal repeat ligated to the bacterial
-galactosidase reporter gene plasmid
(pRSV-lacZ, ATCC) was cotransfected to normalize for DNA transfer
efficiency. For the calcium phosphate method, myocytes (grown on 60-mm
dishes) were incubated with DNA-calcium phosphate solution (containing
10 µg of p3003ANF-luc and 2 µg of pRSV-lacZ) at 37°C for 6 h.
Cells were then washed and maintained in growth medium or in growth
media supplemented with verapamil, PE, or their combination. After an
additional 48 h of culture, the cells were assayed for luciferase and
-galactosidase activities as previously described (25). Relative
light units were measured using an enhanced luciferase assay kit
(Analytical Luminescence Laboratory, Ann Arbor, MI) and a luminometer
(Berthold, model LB9501).
Transfections were also performed with the replication-deficient human
adenovirus type 5 mutant, Ad5dl312, generously provided by Dr. Mary
Kathleen Rundell, Northwestern University, Chicago IL. Ad5dl312
adenovirus was propagated in the complementing human embryonic kidney
cell line HEK-293. Briefly, HEK-293 cells were infected with virus
lysate in a final concentration of 2-5 plaque-forming units/cell
for 29-34 h. The cells were lysed with five cycles of freezing and
thawing to release virus. The virus was then purified by density
gradient centrifugation with two consecutive CsCl gradients (step
gradient 1.2 and 1.45 g/ml). The viral band was collected and dialyzed.
The viral stock solution was stored in aliquots at
70°C.
Virion concentration was determined by optical density (1 optical
density unit at 260 nm = 1012
viral particles). A mixture of 3 × 1010 viral particles, 2.5 µg/ml
poly-L-lysine, 2.5 µg
p3003ANF-luc, and 0.5 µg pRSV-lacZ per milliliter was prepared as
described by Kohout et al. (17). Transfection was initiated by
replacing the culture medium of myocytes (grown on 35-mm dishes) with
500 µl of this transfection mixture per well for 90 min at 37°C.
Transfection was terminated by diluting the transfection mixture by the
addition of 1.5 ml of myocyte growth medium for 14-18 h. Cells
were then maintained in growth medium in the presence or absence of 10 µM verapamil, 50 µM PE, or their combination. After an additional 24 h of culture, the cells were assayed for luciferase and
-galactosidase activities as previously described (25).
ANF mRNA analysis.
Total cellular RNA was isolated by the method of Chomczynski and Sacchi
(7) after 48 h of culture under control conditions or after treatment
with PE, verapamil, or their combination. RNA was quantified by
absorbance at 260 nm, and its integrity was determined by examining the
28S and 18S rRNA bands in ethidium bromide-stained agarose gels. Total
RNA (10 µg) was separated by denaturing agarose gel electrophoresis,
subjected to alkali pretreatment, transferred to nylon membranes by
capillary action, and cross-linked by ultraviolet irradiation. ANF mRNA
levels were detected by hybridization to a
32P-labeled, 786-residue ANF cDNA
probe (20). The Northern blots were also hybridized with a
32P-labeled 24-base
oligodeoxyribonucleotide probe specific for rat 18S rRNA (31).
Data analysis.
Results were expressed as means ± SE. Normality was assessed using
the Kolmogorov-Smirnov test, and homogeneity of variance was assessed
using Levene's test. One-way repeated measures ANOVA, Friedman
repeated measures ANOVA on ranks, or Kruskal-Wallis one-way ANOVA on
ranks followed by the Student-Newman-Keuls test was used for the
statistical comparison of multiple groups, as appropriate. Data were
analyzed using the SigmaStat Statistical Software Package, version 1.0 (Jandel Scientific, San Rafael, CA).
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RESULTS |
PE induces
[Ca2+]i
transients in low-density NRVM cultures.
As seen in Fig. 1, NRVM plated at low
density and maintained in DMEM-medium 199 for 48 h were either
quiescent (Fig. 1A) or displayed
only occasional
[Ca2+]i
transients and contractions (Fig.
1B). However, within 30 min of PE
exposure,
[Ca2+]i
oscillations were induced in >80% of the quiescent myocytes. In the
minority of myocytes that displayed occasional spontaneous [Ca2+]i
transients, PE increased the frequency of
[Ca2+]i
oscillations. Each
[Ca2+]i
transient was accompanied by cell contraction, as assessed by visual
inspection. Pretreatment with verapamil (10 µM) suppressed the
spontaneous
[Ca2+]i
transients and also suppressed the PE-induced
[Ca2+]i
oscillations (Fig. 1C), indicating
that both were highly dependent on
Ca2+ influx via voltage-gated
L-type Ca2+ channels. Both the
frequency and amplitude of the contractile activity appeared to
increase after prolonged exposure to PE. After 24-h exposure, virtually
all of the NRVM were contracting at a rate of 1-2 Hz, whereas NRVM
maintained in medium containing verapamil or PE plus verapamil remained
quiescent.

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Fig. 1.
Phenylephrine (PE) stimulates intracellular
Ca2+ concentration
([Ca2+]i)
transients in low-density neonatal rat ventricular myocyte (NRVM)
cultures. In A,
[Ca2+]i
was recorded from a single fura 2-loaded, quiescent NRVM superfused in
Krebs buffer, followed by Krebs buffer containing 50 µM PE (shaded
bar). In this cell, no spontaneous
[Ca2+]i
transients were noted under control conditions (0-5 min). However,
[Ca2+]i
transients were observed within 1 min after PE exposure. Sampling rate
(1.5 Hz) did not allow for resolution of very rapid
[Ca2+]i
transients that occurred during bursts of activity. In
B, a similar recording was obtained
from a single fura 2-loaded myocyte that displayed occasional
spontaneous
[Ca2+]i
transients. PE increased frequency of
[Ca2+]i
oscillations. In C, a single fura
2-loaded NRVM was first pretreated with verapamil (10 µM, 1 h) and
then superfused with Krebs buffer containing 50 µM PE + 10 µM verapamil. No
[Ca2+]i
transients were generated by PE exposure.
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PE-induced
[Ca2+]i
transients and contractile activity are necessary for hypertrophic
growth.
We next examined whether PE stimulated myocyte growth and whether
PE-induced
[Ca2+]i
transients and contractile activity were required for expression of
specific aspects of the hypertrophic phenotype. NRVM were treated with
PE (50 µM), verapamil (10 µM), or their combination for 48-72 h. Of note, this concentration of verapamil was the minimum
concentration of the drug required to completely inhibit spontaneous
contractile activity over a 24-h period without affecting cell
viability, as assessed by visual inspection for cell detachment.
As seen in Fig.
2A,
PE-treated myocytes were larger, and the increased surface area led to
the formation of more cell-to-cell contacts as compared with untreated,
control cultures. The formation of additional cell-to-cell contacts
over time appeared to increase the number of adjacent myocytes that
were beating synchronously. Although addition of verapamil to the
serum-free culture medium suppressed spontaneous
[Ca2+]i
transients and contractile activity, the myocytes remained well
attached to the collagen substratum. Verapamil only partially blocked
the PE-induced increase in myocyte surface area (Fig. 2B). PE increased myocyte surface
area by 40% in control cultures and by 32% in verapamil-arrested
myocytes.

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Fig. 2.
Effects of PE and verapamil on NRVM cell size. Low-density NRVM
cultures were treated (48 h) with serum-free medium alone (C) or medium
supplemented with PE (50 µM), verapamil (V; 10 µM) or PE and
verapamil (PE + V). Verapamil was added to inhibit both basal
and PE-induced
[Ca2+]i
transients and contractile activity. In
A, live cells were visualized by
Hoffman modulation contrast imaging (all fields viewed with a ×40
objective). In B, cell area
measurements were obtained by fura 2 loading, fluorescence microscopy,
and image analysis using Image-1 software. Data are means ± SE from
120 cells in each group. Data were compared by ANOVA on ranks followed
by Student-Neuman-Keuls test.
* P < 0.05 vs. control. + P < 0.05 vs. verapamil.
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In addition to these morphological changes, PE significantly increased
total protein content in both control and verapamil-treated NRVM (Table
1). However, the PE-induced increase in
total protein content in control cells (27%) was considerably greater
than the increase in total protein observed in cells in which
Ca2+ influx and contractile
activity were blocked by verapamil (16%). PE treatment alone modestly
increased DNA content but substantially increased total protein-to-DNA
ratio as compared with both control and verapamil-treated myocytes.
These results indicate that the PE-induced growth resulted
predominantly from cellular hypertrophy, rather than cellular
hyperplasia. The increases in DNA and total protein-to-DNA ratio were
prevented in NRVM in which Ca2+
influx and contractile activity were blocked with verapamil.
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Table 1.
Inhibition of [Ca2+]i transients and
contractile activity prevents phenylephrineinduced NRVM
hypertrophy
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MHC isoenzyme content was also analyzed in control, PE-treated, and
verapamil-treated cultures. As seen in Fig.
3, control myocytes under these culture
conditions expressed both
-MHC and
-MHC isoenzymes in
approximately equal amounts. Verapamil treatment alone substantially
reduced
-MHC and
-MHC content, which is consistent with the
effects of Ca2+ channel blockade
and contractile arrest on MHC metabolism in high-density NRVM cultures
(3, 31, 32). PE markedly increased the cellular content of both
isoenzymes (
>
) in myocytes maintained in the absence of the
Ca2+ channel blocker. However,
PE-induced
-MHC accumulation was completely prevented when
Ca2+ influx and contractile
activity were blocked with verapamil, whereas
-MHC content was only
modestly increased.
-MHC and
-MHC content from six individual
experiments are summarized in Table 1.

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Fig. 3.
Effects of PE and verapamil on myosin heavy chain (MHC) isoenzyme
content of NRVMs. Low-density NRVM cultures (375,000 cells/35-mm dish)
were treated (48 h) with serum-free medium alone (C) or medium
supplemented with PE (50 µM), verapamil (V; 10 µM), or PE and
verapamil (PE + V) to inhibit PE-induced
[Ca2+]i
transients and contractile activity. Cells were then scraped from
dishes in 500 µl of SDS sample buffer, and protein samples were
separated by SDS-PAGE. Each lane was loaded with 25 µl of cell
extract, except for lane 2 (PE), which
was loaded with 10 µl. Gels were then silver stained and scanned by
laser densitometry. Positions of -MHC and -MHC bands were
confirmed by electrophoresis of known -MHC and -MHC standards
obtained from normal and hypothyroid rat hearts, respectively.
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We also examined the effects of PE and verapamil on sarcomeric
assembly. As seen in Fig. 4, both control
and verapamil-treated myocytes contained few assembled sarcomeres.
Treatment of control, low-density NRVM with PE for 24 h stimulated
sarcomeric assembly, as evidenced by the prominent striated pattern of
actin filaments in FITC-phalloidin-stained cells. Although PE appeared
to increase the amount of filamentous actin staining, PE did not lead
to the appearance of well-formed sarcomeres if
[Ca2+]i
transients and contractile activity were blocked with verapamil. In
keeping with these morphological findings, we found that PE prolonged
the half-life of MHC protein in control but not in verapamil-treated myocytes (Fig. 5). These results are
consistent with previous studies from this laboratory which have
demonstrated that
[Ca2+]i
transients and mechanical activity, in the absence of exogenous agonists, are necessary to maintain sarcomeric assembly and prevent the
accelerated myofibrillar protein degradation associated with contractile arrest (3, 32, 35).

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Fig. 4.
Verapamil blocks PE-induced myofibrillar assembly in NRVM. Low-density
NRVM cultures were maintained (48 h) in serum-free DMEM-medium 199 alone (C) or in medium supplemented with verapamil (V; 10 µM), PE (50 µM), or their combination (PE + V). Cells were then fixed,
permeabilized, stained with FITC-phalloidin, and viewed under a
scanning laser confocal microscope (×60 objective, zoom factor
4.0). As is evident from figure, PE-stimulated sarcomeric assembly was
highly dependent on Ca2+ influx
through voltage-gated L-type Ca2+
channels.
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Fig. 5.
MHC protein half-life in PE-treated NRVM cultures. Low-density NRVM
cultures were biosynthetically labeled (24 h) with
[35S]methionine and
chased (24 h) in serum-free medium alone (C) or medium supplemented
with PE (50 µM), verapamil (V; 10 µM), or their combination (PE + V). Total cell protein was then separated by SDS-PAGE and
autoradiography with fluorographic enhancement. MHC band intensity was
quantified by laser densitometry, and fractional rate of MHC
degradation (MHC
Kd, %/h) for
each condition was estimated by following formula: MHC
Kd = 100 · [ln(MHC
AU)0 ln(MHC
AU)24]/24, where ln(MHC
AU)0 and ln(MHC
AU)24 are natural logarithms of
average absorbance (in arbitrary absorbance units) of MHC bands at
times 0 and 24 h of chase. MHC
Kd values were
converted to apparent half-lives (in h) according to following formula:
MHC t1/2 = 100 · [ln(2)/MHC
Kd]. Data
are means ± SE for 6 myocyte isolations. Data were compared by
repeated measures ANOVA on ranks followed by Student-Neuman-Keuls test.
* P < 0.05 vs. control. + P < 0.05 vs. verapamil.
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|
One potential explanation for the effects of verapamil on PE-induced
myocyte growth was that verapamil directly blocked
1-adrenergic receptor
activation (15), in addition to acting at a site that was downstream of
receptor activation (i.e., the L-type
Ca2+ channel). We therefore
compared the ability of other Ca2+
channel blocking agents (i.e., diltiazem and nifedipine) that do not
exhibit
1-adrenergic receptor
antagonism to suppress PE-induced myocyte hypertrophy. As seen in Fig.
6, equimolar concentrations of nifedipine,
but not diltiazem or verapamil, significantly reduced total
protein-to-DNA ratio in the absence of PE. This reflected the relative
potency of the three Ca2+ channel
blocking agents on the contractile amplitude of spontaneously beating
chick embryo ventricular myocytes (1). However, all three
Ca2+ channel blocking agents
prevented the PE-induced increase in total protein-to-DNA ratio. As
seen in Fig. 7, both diltiazem and
nifedipine also suppressed PE-induced MHC accumulation.

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Fig. 6.
Other Ca2+ channel blockers
prevent PE-induced NRVM hypertrophy. Low-density NRVM cultures were
treated (48 h) with serum-free medium alone (C) or medium supplemented
with verapamil (V; 10 µM), diltiazem (D; 10 µM), or nifedipine (N;
10 µM) in presence or absence of PE (50 µM). To account for
variability in total protein-to-DNA ratio from experiment to
experiment, individual values were expressed as a percentage of control
cells from each myocyte isolation. Data are means ± SE for
4-20 myocyte isolations. Mean ± SE for control group was 52.3 ± 2.8 µg total protein/µg DNA. Data were compared by repeated
measures ANOVA on ranks followed by Student-Neuman-Keuls test.
* P < 0.05 vs. control. + P < 0.05 vs. PE.
P < 0.05 vs. each drug
treatment in absence of PE.
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Fig. 7.
Diltiazem, nifedipine, and 2,3-butanedione monoxime block PE-induced
MHC accumulation in NRVMs. Low-density NRVM cultures were treated (48 h) with serum-free medium alone (C) or medium supplemented with PE (50 µM) or PE and diltiazem (D; 10 µM), nifedipine (N; 10 µM), and
2,3-butanedione monoxime (B; 7.5 mM) to inhibit PE-induced
[Ca2+]i
transients and/or contractile activity. Cells were then scraped
from dishes in 250 µl of SDS sample buffer, and protein samples were
separated by SDS-PAGE. Each lane was loaded with 25 µl cell extract.
Gels were then silver stained and scanned by laser densitometry.
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|
Mechanical activity is required for PE-induced NRVM hypertrophy.
2,3-Butanedione monoxime (BDM), an inhibitor of actin-myosin
cross-bridge cycling, was then used to determine whether mechanical activity, rather than
[Ca2+]i
transients per se, was required to elicit PE-induced NRVM hypertrophy. Previous studies from our laboratory have demonstrated that in NRVM,
acute or chronic exposure to 7.5 mM BDM only modestly reduced the
amplitude of
[Ca2+]i
transients but markedly reduced cell shortening (3, 27). [Higher
concentrations of BDM have been shown to promote voltage-dependent inactivation of L-type Ca2+
channels (11).] Therefore, myocytes were treated (48 h) with PE
(50 µM), 7.5 mM BDM, or their combination, and the resulting cell
extracts were analyzed for total protein, DNA, and MHC content. As seen
in Fig. 8, PE increased total
protein-to-DNA ratio in control, but not in BDM-treated cells.
Similarly, BDM prevented PE-induced
-MHC and
-MHC accumulation
(Fig. 7).

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Fig. 8.
Inhibition of mechanical activity prevents PE-induced NRVM hypertrophy.
Low-density NRVM cultures were treated (48 h) with serum-free medium
alone (C) or medium supplemented with PE (50 µM), 2,3-butanedione
monoxime (B; 7.5 mM), or their combination (PE + B). Data are means ± SE for 6 myocyte isolations. Data were compared by repeated
measures ANOVA on ranks followed by Student-Neuman-Keuls test.
* P < 0.05 vs. control. + P < 0.05 vs. 2,3-butanedione
monoxime.
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|
Phenylephrine activates MAPK independently of
[Ca2+]i
transients and contractile activity.
In contrast to their general effects on cellular growth and
myofibrillar assembly,
[Ca2+]i
transients and contractile activity were not required for PE-induced activation of MAPK. As seen in Fig. 9, both
PMA (200 nM) and PE (50 µM) activated ERK1 and ERK2. MAPK activation
(as assessed by the upward shift in apparent molecular weight of both
ERK1 and ERK2) occurred within 5 min of exposure to either agonist in
control cells, as well as NRVM pretreated (1 h) with verapamil (10 µM) to prevent
[Ca2+]i
transients and beating.

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Fig. 9.
[Ca2+]i
transients and contractile activity are not required for
mitogen-activated protein kinase activation by PE. Low-density NRVM
cultures were maintained in myocyte growth medium for 48 h. Cells were
then switched to fresh growth medium or growth medium containing
verapamil (10 µM). After an additional 1 h, myocytes were stimulated
(5 min) with phorbol 12-myristate 13-acetate (PMA; 200 nM) or PE (50 µM). Total protein homogenates (25 µg) were separated by SDS-PAGE
and transferred to nitrocellulose membrane. Blots were probed with a
mixture of polyclonal antibodies directed toward ERK1 (44 kDa) and ERK2
(42 kDa). Apparent molecular masses are indicated to
left of bands. Activation of ERK1 and
ERK2 is represented by upward shift in molecular mass, designated as
p44 and p42, respectively.
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Effects of
[Ca2+]i
transients, contractile activity, and PE on ANF gene expression.
We also examined the role of
[Ca2+]i
transients and contractile activity in PE-induced stimulation of ANF
promoter activity. As seen in Fig.
10A in
which the CaPO4 method was used in
transient transfection experiments, normalized luciferase activity in
PE-treated myocytes was 246 ± 19%, where expression in control
cells for each of six experiments was normalized to 100%. Addition of
verapamil to the serum-free culture medium significantly reduced basal
ANF promoter activity to 20 ± 1% of control cells. However, PE was still capable of stimulating ANF promoter activity even in the presence
of the Ca2+ channel blocking agent
(38 ± 2% of control cells, or nearly double the level of
expression as compared with cells treated with verapamil).

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Fig. 10.
Effects of
[Ca2+]i
transients, contractile activity, and PE on atrial natriuretic factor
promoter activity. In A, NRVM were
transiently transfected with p3003ANF-luc and pRSV-lacZ using
CaPO4 method. Cells were then
maintained in control medium (C) or medium supplemented with verapamil
(V; 10 µM), PE (50 µM), or their combination. After an additional
48 h of culture, cell extracts were assayed for luciferase and
-galactosidase ( -gal) activities. Data were expressed as
luciferase/ -galactosidase activity where enzyme activity in control
cells was normalized to 100%. Normalized luciferase activity was 4.4 ± 1.1 × 107 relative
light units in control cells. Data are means ± SE from 6 different
cell isolations. In B, transfections
with p3003ANF-luc and pRSV-lacZ were also performed with
replication-deficient adenovirus Ad5dl312. Cells were then maintained
in control medium (C) or medium supplemented with verapamil (10 µM),
PE (50 µM), or their combination. After an additional 24 h of
culture, cell extracts were assayed for luciferase and
-galactosidase activities, as described above. Normalized luciferase
activity was 1.3 ± 0.7 × 105 relative light units in
control cells. Data are means ± SE from 4 different cell
isolations. Data were compared by repeated measures ANOVA on ranks
followed by Student-Neuman-Keuls test.
* P < 0.05 vs. control.
+ P < 0.05 vs. verapamil.
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|
Because of the relatively low transfection efficiency using the
CaPO4 method, we also examined the
effects of PE, verapamil, and their combination on ANF promoter
activity using adenoviral-assisted transfection. This technique
produces much higher levels of transfection efficiency in NRVM (17).
After cotransfection of p3003ANF-luc and pRSV-lacZ, luciferase and
-galactosidase activities were then assayed 24 h after exposure to
PE, verapamil, or their combination. As seen in Fig.
10B, normalized luciferase activity in
PE-treated myocytes was 206 ± 24%, where expression in control
cells for each of four experiments was normalized to 100%. Addition of
verapamil to the serum-free culture medium again significantly reduced
basal ANF promoter activity to 40 ± 3% of control cells. However,
PE was again still capable of stimulating ANF promoter activity even in
the presence of verapamil (1.7-fold increase over cells treated with
verapamil alone). Thus both transfection techniques demonstrated [Ca2+]i/contraction-dependent
and
[Ca2+]i/contraction-independent
components of PE-stimulated ANF promoter activity.
Finally, we examined whether the observed changes in ANF promoter
activity corresponded to similar alterations in endogenous ANF mRNA
levels. As seen in Fig. 11, exposure of
NRVM to PE for 48 h increased ANF mRNA levels in both control and
verapamil-treated myocytes.

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Fig. 11.
Effects of
[Ca2+]i
transients, contractile activity, and PE on atrial natriuretic factor
(ANF) mRNA levels. NRVM were maintained (48 h) in control medium (C) or
medium supplemented with PE (50 µM), verapamil (V; 10 µM), or their
combination (PE + V). Total RNA (10 µg) was size fractionated,
transferred to nitrocellulose, and sequentially probed with
32P-labeled cDNA and
oligodeoxyribonucleic acid probes specific for rat ANF mRNA and 18S
rRNA, respectively. As is evident from figure, PE increased ANF mRNA
levels in both control and verapamil-treated NRVM. Similar results were
obtained in 2 other experiments.
|
|
 |
DISCUSSION |
Cultured NRVM have been extensively used as a model system with which
to define the signal transduction pathways that cause hypertrophic
growth of cardiac muscle in response to both neurohormonal and
mechanical stimuli. With respect to the growth-promoting effects of
mechanical load, several studies have shown that NRVM undergo hypertrophy in response to either externally applied or intrinsically generated mechanical load in the absence of exogenous growth factors. However, there are several potential mechanisms wherein neurohormonal and mechanical signaling pathways may interact to elicit specific aspects of the hypertrophic phenotype. For instance, isolated NRVM
plated at low density onto a collagen-coated substratum exhibit little
spontaneous contractile activity. However, Simpson (38) showed that the
catecholamines norepinephrine (NE) and epinephrine (E) stimulated
contractile activity in quiescent, low-density NRVM cultures maintained
in serum-free medium. The effects of NE and E on contractile frequency
appeared to be due to the ability of the drugs to simultaneously
stimulate both
1- and
1-adrenoreceptors.
1-Adrenergic stimulation alone
reportedly produced enlarged cells that did not beat, whereas combined
1- and
1-adrenergic stimulation slowly
induced contractile activity over a 24-h period even when protein
synthesis and hypertrophy were inhibited with cycloheximide (39). In
contrast, Kimura et al. (16) showed that NE evoked a positive
chronotropic response within 1 min of exposure to the drug. This rapid
induction of beating was dependent only on activation of
1-adrenoreceptors, since
contractile activity was induced by exposure to NE in the presence of
the
-receptor antagonist propranolol. Our present results depicted
in Fig. 1 are consistent with the observations of Kimura et al. (16)
and indicate that in cultured NRVM,
1-adrenoreceptor stimulation
triggers
[Ca2+]i
transients that are highly dependent on voltage-gated L-type Ca2+ channels. It is also apparent
from Fig. 1 that PE does not appreciably stimulate inositol
1,4,5-trisphosphate-mediated Ca2+
release from intracellular stores, since no
[Ca2+]i
increase was detected when Ca2+
influx was blocked with verapamil.
PE and other Gq-coupled agonists
have been shown to also trigger a variety of signaling events
associated with the hypertrophic phenotype. These phenotypic
alterations include the tyrosine phosphorylation of several signaling
molecules including ERK1 and ERK2, induction of
c-fos and other immediate/early genes,
transcriptional activation of the secondary response genes
-MHC,
-skeletal actin and ANF, increased protein synthesis, and the
assembly of newly synthesized myofibrillar proteins into sarcomeres.
However, the role of
[Ca2+]i
transients and mechanical activity in these signaling events remains
controversial. In the present study, we have attempted to dissociate
features of PE-stimulated myocyte hypertrophy that are dependent on
[Ca2+]i
transients and contractile activity from those that do not require this
activity.
As reviewed by Chien et al. (6), induction of the myocyte hypertrophic
phenotype by a variety of extracellular signals requires both
transcriptional activation and enhanced assembly of individual
contractile protein subunits into sarcomeres. Our results clearly
indicate that
[Ca2+]i
transients and mechanical activity are critical for the alterations in
cellular architecture that are typical of PE-induced NRVM hypertrophy. Despite a careful examination of several signal transduction pathways, the intracellular mechanisms responsible for PE-induced sarcomeric protein assembly remain largely unknown. Initial studies implicated protein kinase C (PKC) activation in sarcomeric protein assembly. Dunmon et al. (9) showed that PKC activation of low-density NRVM
cultures with either PE or the phorbol ester PMA not only induced
immediate early gene expression and stimulated nuclear gene
transcription, but also caused the appearance within the cytoplasm of
organized sarcomeres. Other protein kinases, including Raf-1, MAPK
kinase (MAPKK or MEK), MAPK, and S6 kinase were also activated by PE
treatment or PMA (for review, see Ref. 2). However, activation of PKC
with PMA did not stimulate myofibrillar assembly when
Ca2+ influx was blocked by the
L-type Ca2+ channel blocker
verapamil, despite a marked degree of PKC activation/translocation (32, 35; unpublished data). Furthermore, the slow assembly of myosin light
chain-2 into sarcomeres in response to PE treatment was not dependent
on activation of MAPK (41), despite the fact that this kinase has been
implicated in the transcriptional activation of numerous cellular genes
essential for growth. As indicated in Fig. 4 of the present study,
PE-induced sarcomeric assembly required the induction of
[Ca2+]i
transients and contractile activity. These results support previous
studies which indicate an important role for intrinsic and/or
externally applied mechanical load in the induction and maintenance of
myofibrillar protein assembly (3, 8, 31, 35-37). As demonstrated
here and in previous studies, the assembly of myofibrillar proteins
such as MHC and actin into functional sarcomeres also profoundly
reduced the susceptibility of these proteins to intracellular
degradation, thereby contributing to contractile protein accumulation
and cellular hypertrophy (3, 8, 32, 35-37).
One mechanism whereby PE-induced
[Ca2+]i
transients and mechanical activity may stimulate sarcomeric assembly in
cultured NRVM is by the load-dependent formation of focal adhesions and
costameres. In a recent study, Sharp et al. (34) demonstrated that both intrinsic and externally applied mechanical load stabilized the cell-surface distribution of
1-integrin, the transmembrane
cell surface receptor which mediates cell attachment to the
extracellular matrix. Inhibition of the spontaneous contractile
activity of high-density NRVM with nifedipine caused the rapid
disruption of focal adhesions and the loss of
1-integrin from the cell
surface, whereas application of 5% static stretch partially prevented
these changes. Restoration of contractile activity (by removal of
nifedipine from the culture medium) caused the reaccumulation of
1-integrin on the cell surface
and the reformation of focal adhesions which temporally corresponded
with the reassembly of myofibrillar proteins into sarcomeres. Thus
[Ca2+]i
transients and actin-myosin cross-bridge formation (even in the absence
of other agonists) were sufficient for sarcomeric assembly (3, 24, 31).
Nevertheless, nonsarcomeric, filamentous actin appeared to accumulate
in PE-stimulated NRVM even in the presence of verapamil (Fig. 4), which
may explain why the PE-stimulated increase in cell surface area was
relatively resistant to blockade of
Ca2+ influx.
In contrast to the clear dependence of sarcomeric assembly on
PE-stimulated
[Ca2+]i
transients and mechanical activity, we found that MAPK activation and
ANF gene expression were less dependent on these signals. Our results
should be evaluated in light of prior studies by Sadoshima et al. (29),
which suggested that ANG II-mediated activation of MAPK was highly
dependent on
[Ca2+]i.
Their conclusion was based on the finding that pretreatment of NRVM
with
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM blocked the ANG II-mediated increase in resting [Ca2+]i
and also completely prevented agonist-induced MAPK activation. However,
it should be pointed out that ANG II did not stimulate [Ca2+]i
oscillations in their low-density cultured heart cells. Furthermore, the concentration of BAPTA-AM used to prevent the ANG II-induced increase in
[Ca2+]i
actually lowered resting levels of
[Ca2+]i.
As indicated in Figs. 1 and 10 of this report, verapamil treatment did
not significantly lower resting
[Ca2+]i
or block MAPK activation but clearly suppressed phenylephrine-induced [Ca2+]i
transients and beating. These results are in agreement with previous
studies that have indicated that MAPK activation occurs by both
Ca2+-dependent and
Ca2+-independent pathways (5, 19).
Although verapamil treatment decreased basal ANF promoter activity
(Fig. 10) and ANF mRNA levels (Fig. 11),
1-adrenergic stimulation was
still capable of augmenting ANF gene expression in the absence of
[Ca2+]i
transients and contractile activity. Our results confirm and extend
previous studies by Sei et al. (33), who demonstrated that
1-adrenergic stimulation
significantly increased ANF mRNA levels in both control and
nifedipine-treated myocytes. Thus the analysis of ANF promoter activity
by transient transfection, as well as by quantitative Northern
blotting, revealed
[Ca2+]i/contraction-dependent
and
[Ca2+]i/contraction-independent
components of PE-stimulated ANF gene expression. Furthermore, our
present finding of decreased basal ANF transcription in
verapamil-treated cells suppports previous studies from this laboratory
using spontaneously contracting, high-density NRVM (10). Those studies
demonstrated that contractile arrest (produced with verapamil, BDM, or
K+ depolarization) markedly
reduced basal ANF promoter activity, mRNA levels, and protein secretion
(10).
In summary,
1-adrenergic
stimulation induced
[Ca2+]i
transients and contractile activity that were required for MHC
accumulation and sarcomeric assembly in cultured NRVM. The
intracellular mechanisms responsible for generating
[Ca2+]i
oscillations in response to
1-adrenergic stimulation in
these cultured heart cells requires further investigation, but the
results of the present report highlight the importance of both
mechanical and neurohormonal factors (and their potential interactions)
in eliciting specific aspects of the hypertrophic phenotype.
 |
ACKNOWLEDGEMENTS |
We thank M. Lisa Spragia and Alan G. Ferguson for excellent
technical assistance and Peggy Richied for help in preparation of the
manuscript.
 |
FOOTNOTES |
These studies were supported by National Heart, Lung, and Blood
Institute (NHLBI) Grants RO1-HL-34328 and HL-52478 and by gifts to the
Cardiovascular Institute from the Nalco Foundation, the Eugene J. and
Elsie E. Weyler Endowment for Clinical Cardiology Research, and the
Ralph and Marian Falk Trust for Medical Research. D. M. Eble was a
recipient of NHLBI National Research Service Award F32-HL-09611 during
the time these studies were performed.
Address for reprint requests: A. M. Samarel, Loyola University Medical
Center, Bldg. 110, Rm. 5222, 2160 South First Ave., Maywood, IL 60153.
Received 2 September 1997; accepted in final form 22 January 1998.
 |
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