Download Contraction Modulates the Capacity for Protein Synthesis During

542
Contraction Modulates the Capacity for
Protein Synthesis During Growth of Neonatal
Heart Cells in Culture
Paul J. McDermott and Howard E. Morgan
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Neonatal ventricular myocytes that were incubated in a well-defined serum-free medium
containing 50 mM KC1 did not contract and maintained stable cell size, as assessed by the
protein/DNA ratio. The present study utilized KCl-arrested cells to examine the effect of
constant rates of synchronous contraction in normal [K + ] o (4 mM) as a physiological stimulus
for myocyte growth. Cell growth increased following the onset of contraction when measured
over 3 days. The rate of protein synthesis was accelerated in parallel by contraction, but the rate
of protein degradation remained similar to rates in noncontracting cells. The capacity for
protein synthesis was estimated by total RNA content and was increased in contracting as
compared with KCl-arrested cells. This increase was accompanied by faster rates of RNA
synthesis as determined from the incorporation of [3H]uridine into RNA and the specific activity
of the cellular UTP pool. The rate of RNA degradation was accelerated during contraction but
the difference between the rates of RNA synthesis and degradation resulted in net RNA
accumulation of 49% after 3 days. These data demonstrated that 1) contractile activity
stimulated myocyte growth through an increased capacity for protein synthesis and 2) the
increased capacity for protein synthesis involved acceleration of the rate of RNA synthesis. Since
enhancement of protein synthetic capacity is a common feature of myocyte hypertrophy in vivo
and in vitro, this model can be used to examine the regulation of ribosome synthesis during
hypertrophic growth. (Circulation Research 1989;64:542-553)
C
ultured myocytes, and particularly cells from
neonatal rats, have proven to be useful for
studies of myocardial growth and metabolism.12 Growth of these cells occurs primarily by
hypertrophy, rather than cell division,2 and is accompanied by changes in morphology,3-4 ultrastructure,5
and the cellular content and expression of contractile
protein isoforms such as myosin and actin.6-7 When
neonatal myocytes are maintained in chemically
defined media, protein synthesis can be modulated in
response to hormone treatment.8-10 Chronic administration of norepinephrine accelerates the rate of
protein synthesis and, in parallel, myocyte growth.10
The hypertrophic response to norepinephrine is
induced by binding to aradrenergic receptors on the
cell surface, which generate intracellular signals that
From the Sigfried and Janet Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania, and the Department of
Physiology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania.
Supported by National Institutes of Health Grant HL-35289.
PJ.M. was the recipient of a National Research Service Award
HL-07223.
Address for correspondence: Dr. Howard E. Morgan, Weis
Center for Research, Geisinger Clinic, Danville, PA 17822.
Received June 28, 1988; accepted August 18, 1988.
increase growth through as yet undetermined
mechanisms." The stimulation of cell growth in
response to norepinephrine occurs independent of
adrenergic enhancement of contractile activity.12 In
contrast, recent studies have demonstrated that regular rates of contraction in low KC1 medium stimulate growth of adherent neonatal myocytes in
culture.13 In general, the specific mechanisms by
which protein synthesis is regulated at either the
transcriptional or translational level have not been
defined for any growth stimulus.
Faster rates of protein synthesis during hypertrophic growth could potentially result from either
an increased efficiency of protein synthesis or greater
capacity for protein synthesis or both.14 In cardiac
muscle, an increased capacity for protein synthesis
reflects ribosome content and appears to be the
primary mechanism for sustained growth. This conclusion is supported by two observations. First, a
large percentage of ribosomes (88-90%) are present
in the form of polysomes in nongrowing cardiac
muscle.15 This finding suggests that the rate of
peptide chain initiation and the availability of components of the translation pathway, including
mRNA, do not limit growth. Secondly, greater
McDermott and Morgan
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tissue content of RNA14 and faster rates of RNA
synthesis16 and ribosome formation1417 are found in
growing hearts in response to a variety of stimuli. A
similar mechanism may exist in vitro since RNA
content increases during myocyte hypertrophy.7
However, changes in the efficiency of protein synthesis may serve as an alternative mechanism for
initiating hypertrophy particularly as an acute
response to changes in workload.18
The present study was undertaken to test the
hypothesis that hypertrophic growth of myocytes in
culture is modulated by changes in the capacity for
protein synthesis. To test the hypothesis, RNA
content in synchronously contracting myocytes was
compared with noncontracting cells arrested by the
addition of 50 mM KC1. Potassium chloride-arrested
myocytes proved to be a suitable model that
responded to the onset of synchronous contractile
activity with continuous hypertrophic growth and
faster rates of RNA and protein synthesis. The rate
of protein synthesis correlated with the content of
RNA, in both spontaneously contracting and KC1arrested myocytes. This model was used to assess
the relative contributions of RNA synthesis and
degradation in modulating RNA content during
growth induced by contraction.
Materials and Methods
Cell Cultures and Media
Primary cell cultures were prepared from minced
ventricular myocardium of 2-4-day-old neonatal
rats with minor modifications, as described previously.1-6 The cells were dissociated in a water
jacketed Celstir apparatus (Wheaton Scientific, Millvale, New Jersey) at 37° C with a mixture of partially purified trypsin (Cooper Biomedical, Malvern, Pennsylvania), chymotrypsin, and elastase
(Sigma Chemical Co, St. Louis, Missouri). The
concentration of enzymes were 2.4 IU/ml, 2.7 IU/
ml and 0.94 IU/ml respectively, and the proteins
were dissolved in Ca2+-Mg2+ free Hanks' salt solution buffered with 30 mM HEPES (/V-2-hydroxyethylpiperazine-W-2-ethanesulfonic acid), pH 7.4.
After each of six successive 20-minute incubations,
the dissociated cells were mixed with minimum
essential medium Eagle (MEM) (GIBCO, Grand
Island, New York) containing 10% newborn calf
serum and were centrifuged and pooled. The dissociated cells were enriched for cardiomyocytes by
the technique of differential adhesion for 1.5 hours
and plated at a concentration of approximately
4x 10s cells/60 mm dish. After an overnight incubation in MEM containing 10% newborn calf serum
and 0.1 mM 5-bromo-2'deoxyuridine (BrdU, Sigma),
the attached cells were rinsed and maintained in
serum-free medium.19 Briefly, standard MEM was
supplemented with MEM amino acids, vitamins,
penicillin-streptomycin (GIBCO), and 2 mM glutamine. In addition, the medium contained 30 nM
NaSeO4, 10 fig human transferrin/ml (Sigma), 0.1
Protein Synthesis in Cultured Heart Cells
543
mM BrdU, and, as a substitute for gluconolactone,
0.25 mM ascorbic acid. High (K+) medium was
prepared by adding a stock solution of KC1 in MEM
to achieve a final concentration of 50 mM. The
following radioactive precursors were added to the
culture medium as indicated: [5,6-3H]uridine (ICN,
Irvine, California; 42-50 Ci/mmol, 25 ^tCi/ml), [2,63
H]phenylalanine (Amersham, Arlington Heights,
Illinois; 50-58 Ci/mmol, 25 /iCi/ml, or 2.5 fiCi/ml),
and phenylalanine I4C (ul) (Amersham; 5 mCi/mmol,
0.1 /xCi/ml).
DNA Content
The total DNA content per culture dish was
determined after washing the cells three times in
ice-cold phosphate-buffered saline (PBS) and scraping the cells into sodium citrate buffer containing
0.25% (wt/vol) sodium dodecyl sulfate and freezing
at -20° C. Prior to assay, the homogenates were
thawed and vortexed extensively, and DNA was
measured fluorometrically using calf thymus DNA
as a standard.20 In some experiments, cell samples
were precipitated in 0.5N HC104 and used to determine RNA content as described below.
Protein Synthesis
Rates of protein synthesis were determined by two
methods. In some experiments, cultures were pulselabeled with [3H]phenylalanine (PHE), 25 /iCi/ml, in
culture medium which contained 0.4 mM phenylalanine. To determine the specific radioactivity of labeled
protein, the cells were rinsed in PBS and precipitated
with 10% trichloroacetic acid (TCA). The pellets
were boiled for 10 min in 10% TCA containing 1 g
PHE/1, solubilized in 0.3N NaOH for 1 hour (37° C),
and reprecipitated by adding hydrogen chloride to a
concentration of 1.5N. The pellets were dried by
successive washes in absolute ethanol; ethanol, chloroform, ether (3:2:1); and ether. The pellets were
solubilized in 0.3N NaOH. Radioactivity and protein
concentrations21 were measured and specific radioactivities calculated.
Specific radioactivity of transfer RNA (tRNA)bound PHE and PHE in the culture media were
determined by the method of Airhart et al22 except
that phenol was added directly to unrinsed cells in
the culture dishes after the media were removed.
After phenol extraction and ethanol precipitation,
the RNA pellet was washed extensively with ethanol (five times) before deacylation of tRNA-bound
PHE. [3H]PHE was reacted with 2.5 mM [Nmethyl-MC]dansyl-Cl (dimethylaminonaphthalene5-sulphonyl chloride) (Amersham; 50-70 mCi/mmol,
120-150 dpm/pmol). In control experiments, contamination of the RNA pellet with [3H]PHE
amounted to no more than 0.1 pmol, a quantity
20-fold less than PHE derived from tRNA. Protein
synthesis was calculated by the following formula:
[protein specific radioactivity (dpm/mg)]/[PHE specific radioactivity (dpm/nmol)]/hr.
544
Circulation Research
Vol 64, No 3, March 1989
In other experiments, the fractional rate of protein synthesis (KJ was determined.23 Briefly, cells
were labeled to equilibrium with [14C]PHE (0.1
ml) and pulse-labeled for 4 hours [3H]PHE (2.5
ml). In agreement with Meidell et al10, the specific
radioactivity of total protein was constant after 4
days of continuous labeling, indicating that the
specific radioactivities of medium and protein
[MC]PHE were equilibrated (data not shown). Incorporation of [3H]PHE was linear over 4 hours in
equilibrium-labeled cells (data not shown). Fractional rates (KJ were calculated by the formula
K,=-
P*(t2)-P*(t.)
- ^ P*(t)dt
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where P* and F* are the specific activities at times t,
and t2 in protein and precursor respectively.
The specific radioactivity of intracellular PHE
was determined by high pressure reverse phase
liquid chromatography using phenylisothiocyanate
(PITC)-derivatized amino acids (Pierce Chemical
Co, Rockford, Illinois). Cells were washed extensively in PBS and precipitated with 95% ethanol,
and the soluble material was dried for PITC
derivatization.24 PITC-PHE was resolved with a 4.6
mmx25 cm ultrasphere-ODS column (Beckman,
San Ramon, California) using 0.1 M sodium acetate
in the mobile phase and a step gradient of acetonitrile (6%-12%-24%). The quantity of PHE was
calculated from peak areas using PHE standards by
monitoring of absorbance at 260 mM. Specific radioactivities for [3H]PHE in the culture medium were
determined by the same procedure.
Analysis of Percentage ofRNA
in Ribosomal Subunits
The percentage of RNA in ribosomal subunits
was determined by the method of Morgan et al25
with minor modifications. Cells from five culture
dishes were rinsed three times in ice-cold PBS and
scraped in a buffer containing 0.25 M KC1, 2 mM
MgCl2, and 10 mM Tris, pH 7.4. The cells were
homogenized in a Dounce homogenizer, treated
with Triton X-100 (0.3%) and centrifuged at 10,000#
for 10 minutes. An aliquot of the postmitochondrial
supernatant was layered on 15-68% exponential
sucrose gradients in the same buffer and centrifuged
for 15 hours at 40,000g. The gradients were fractionated (ISCO, Lincoln, Nebraska), and the 40S
and 60S peaks were collected and used to measure
RNA. The total amount of RNA in an aliquot of the
supernatant of the homogenate was determined and
used to calculate the fraction of total RNA in
ribosomal subunits. The recovery of RNA in the
postmitochondrial supernatant was not affected by
preincubation of cells in medium containing 0.13
mM puromycin. This concentration inhibited peptide chain elongation in rat heart.13
RNA Content and RNA Synthesis
RNA content per culture dish was measured by
the method of Munro and Fleck.26 Each dish was
rinsed three times in PBS, and the cells were
scraped into 0.5N HC1O4. The dish was rinsed twice
with acid. The extract was centrifuged, and the
pellet was washed three times in 0.5N HCIO* before
incubation in 0.3N NaOH for 1 hour at 37° C. The
protein was reprecipitated by the addition of 4N
HCIO* to a final concentration of IN. After centrifugation, absorbance at 260 run was measured, and
the RNA concentration per dish was calculated as
described.15 The radioactivity of RNA was determined by liquid scintillation counting, and the specific radioactivity was calculated.
The rate of RNA synthesis (pmol UMP incorporated/jig RNA/hr) was calculated as follows: [RNA
specific radioactivity (dpm/pig)]/[UTP specific radioactivity (dpm/pmol)]/hr. UTP specific radioactivity
for each dish was determined using HCIO4 extracts
of whole cells. The HC104 extracts were neutralized
by addition of 2 M KHCO3 to a final concentration
of 0.5 M, the perchlorate was removed by centrifugation, and the supernatant filtered through 0.2fjbm filters (MilJipore Corp, Bedford, Massachusetts). The filtrates were concentrated in a vacuum
centrifuge (Savant Instruments, Inc, Farmingdale,
New York) before purification of UTP by high
pressure liquid chromatography (Beckman) using a
4 mmx25 cm Bio-Sil ODS-5S column (Bio-Rad
Laboratories, Richmond, California). UTP was purified by the method of Folley et al27 with a buffer of
30 mM H3PO4-triethylamine, pH 6.5, and a gradient
of 80 /u.M MgSO4/min. Ultraviolet absorbance was
monitored at 260 nm. The areas under the peak
were integrated and compared with known standards. Fractions corresponding to the retention
time of UTP were used to determine radioactivity.
Specific radioactivity of [3H]uridine in the culture
medium was measured in HC1O4 extracts that were
neutralized and filtered before purification by highperformance liquid chromatography, as described
above.
Statistical Analyses
Comparisons between two means were performed
using Student's t test. A value of p^0.05 was taken
to indicate a significant difference. Values are reported
as mean±SEM, and the number of samples are
indicated in the tables and figure legends.
Results
Culture of KCl-Arrested Myocytes
Previous studies have demonstrated that noncontracting cells arrested by chronic administration of
50 mM KC1 maintain the ability to grow and develop
myofibrils.3 Approximately 85% of the cells were
myocytes as determined by immunofluorescent staining with antimyosin antibodies, and the number of
myocytes per culture dish did not change during
McDermott and Morgan
30
Protein Synthesis in Cultured Heart Cells
545
r
-i 1-0
<
z
a
z
z
a
uj
O
a.
a.
20
0.5
_i
O
10
0.1
r
I
2
-i
<
FIGURE 1. Cell growth and RNA content in
KCl-arrested myocytes. Cells were incubated,
as described in "Materials and Methods," in
serum-free medium to which 50 mM KCl was
added beginning on day I. The ratio of total cell
proteinlDNA (•) and total RNAIDNA (o) was
measured at daily intervals. Values are the
mean±SEM for at least nine culture dishes.
I
3
4
5
6
TIME IN CULTURE (DAYS)
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KCl arrest.28 Myocytes were maintained in a serumfree medium that contained 0.1 mM BrdU throughout the entire course of each experiment to prevent
proliferation of nonmyocytic cells. When total protein/DNA ratios were used as an index of cell
growth, growth increased for the first 2 days (Figure
1). Since absolute values for DNA did not change
significantly during the first 2 days of KCl arrest
(47.8 /xg DNA/dish, day 1; 47.1 fig DNA/dish, day
3), this period of rapid growth was due to net
protein accumulation. From 3 days onward, cell
growth ceased and RNA content was unchanged.
The cessation of growth observed after 2 days of
KCl arrest was not the consequence of plating
density because cell growth was observed in cultures that contained approximately 60 ^.g DNA per
culture dish, a value 50% above the usual plating
density. In earlier studies,29 cells were plated at a
concentration of 5 x 10* cells/60 mm dish. This density did not inhibit growth of neonatal myocytes
maintained in serum-supplemented media. A decline
in DNA content per culture dish that averaged 9.6%
(five preparations) was observed between days 3
and 6 of KCl arrest. However, this decrease was
variable and was accompanied by a proportional fall
in cell protein. Previous studies have demonstrated
that the addition of 0.1 mM BrdU selectively suppressed the proliferation of nonmuscle cells and
resulted in a small decrease in cell number.4 This
decrease was almost entirely due to a loss of
nonmuscle cells.4 These findings indicated the KClarrested cells were a suitable experimental system
in which to study the effects of resumption of
contractile activity on hypertrophic growth.
Effects of Contraction on Protein Synthesis
The next series of experiments were undertaken
to determine whether a decline in the rate of total
protein synthesis accounted for the cessation of
growth in arrested myocytes. An accurate determination of the rate of protein synthesis required that
the specific radioactivity of the precursor pool of
PHE be used to calculate synthesis rates. Specific
radioactivity of tRNA-bound [3H]PHE was measured and compared with the specific radioactivity
of [3H]PHE in the culture medium. The final PHE
concentration in the culture medium was 0.4 mM, a
concentration that resulted in equilibration of the
specific radioactivity of perfusate and tRNA-bound
PH]PHE in rat h e a r t s The specific radioactivity of
tRNA-bound [3H]PHE was constant over 2 hours of
incubation (Table 1) but was approximately 53-59%
of the specific radioactivity of PHE in the culture
medium. This extent of equilibration was similar to
that observed for cultured lung macrophages radiolabeled with PHE and cultured chick myocytes or
skeletal muscle cells radiolabeled with leucine.3132
The relation of the extracellular and intracellular
PHE pools was further evaluated by comparison of
the specific radioactivities of intracellular free
[3H]PHE with that in the culture medium (Figure 2).
The specific radioactivities of intracellular and
medium PHE were equilibrated after 5 minutes and
remained so over 2 hours of labeling. The incorporation of [3H]PHE into total protein was linear.
These findings demonstrated that charging of
phenylalanyl-tRNA did not occur from a wellmixed intracellular pool despite the fact that the
specific radioactivities of medium and intracellular
TABLE 1. Spedfic Radioactivities of Medium and tRNA-Bound
[3H]PHE During Pube Labeling
[3H]PHE specific radioactivity
(dpm/nmol)
Medium
tRNA-bound
96,996±4,808 (12)
30 minutes
51,864±12,044*(3)
60 minutes
120 minutes
53,796+7,463* (4)
57,561 ±8,549* (4)
Specific radioactivities of medium and tRNA-bound phenylalanine (PHE) were measured by the dansyl-CI method as described
in "Materials and Methods."
*p<0.005 versus specific radioactivity of medium PHE.
546
Circulation Research
Vol 64, No 3, March 1989
1000
o
<
o
u
u.
150O
~
O
a.
o
E
E
o
u
i
100
10-
2.
30
tO
a
x f
E
FIGURE 2. Specific radioactivities of
medium and intracellular phenylalanine
(PHE) and PHE incorporation. Specific
radioactivities of medium (o) and intracellular (•) PHE were measured as described
in "Materials and Methods." Incorporation of [3H]PHE into total cell protein is
shown by the triangles. Results are from a
single preparation, but similar results were
obtained in an identical experiment.
120
INCUBATION TIME (MINUTES)
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[3H]PHE were equilibrated. There appeared to be
preferential reutilization of PHE derived from protein degradation. Therefore, unless the specific radioactivity of tRNA-bound PHE had been measured
and used as the immediate precursor in calculations
of protein synthesis (Table 1), rates would have
been underestimated by approximately 35%. This
approach to the measurement of the rate of protein
synthesis was further validated by measuring K, by
the dual isotope method. The values for Ks as
measured using dual isotopes were 0.01% and 0.0191
per hour for days 5 and 6 during KC1 arrest respectively (Table 2). These values were compared with
fractional rates of total protein synthesis calculated
by dividing the rates of total protein synthesis at
days 5 and 6 during KC1 arrest (Figure 3) by the
PHE content of total heart protein. The PHE content (280 nmol/mg protein) observed in protein from
total heart and cultured lung macrophages was used
in this calculation.3133 The fractional rates derived
from the rates of protein synthesis in Figure 3 were
approximately 0.021 and 0.020 per hour at days 5
and 6 during KC1 arrest, respectively. These rates
were in good agreement with those obtained using
TABLE 2.
Effects of Contraction on Accumulation of Protein and RNA and Protein and RNA Synthesis
Onset of
contraction
(days)
0
Protein: DNA ratio
(g protein/g DNA)
RNA:DNA ratio
(g RNA/g DNA)
21.1 + 1.2
0.75±0.04
(23)
(17)
1
2
3
dual isotopes. Rates of total protein synthesis were
faster during the first 3 days in culture (Figure 3) but
decelerated and stabilized at the time cell growth
essentially ceased.
When myocytes maintained in 50 mM KC1 were
switched to normal KC1 medium (4 mM) after 2
days of KC1 arrest, spontaneous, synchronous contraction resumed within approximately 0.5-1 hour.
Arrested and beating cells were used to study the
effect of contractile activity on cell growth and
protein synthesis. The protein/DNA ratios of contracting and KCl-arrested myocytes were measured
at daily intervals following the onset of contraction.
Protein/DNA ratios increased significantly after 2
and 3 days of contractile activity but did not change
in KCl-arrested cells (Table 2). Fractional rates of
protein synthesis were compared in contracting and
KCl-arrested cells. Measurements of K, required
that [I4C]PHE equilibrium-labeled myocytes were
in a steady state; that is, rates of protein synthesis
and degradation were equal, prior to pulse labeling
with [3H]PHE. Steady-state conditions for cell
growth were not achieved in contracting cells, and,
as a result, apparent K, values were obtained. After
Arrested
Contracting
Arrested
Contracting
22.7±1.7
(17)
23.4±1.0
(20)
23.3±1.0
(13)
25.3±2.3
(10)
29.2±3.1*
(11)
29.9±2.2*
(8)
0.74±0.03
(26)
0.77±0.04
(27)
0.69+0.03
0.98+0.05*
(19)
1.12±0.06*
(21)
1.12±0.O4*
(14)
(27)
Fractional rate of
protein synthesis (K.J
Fractional rate of
RNA synthesis (K J
O.O363±O.OO25
(4)
Arrested
0.0196±0.0014
(4)
0.0191 ±0.0016
(5)
Contracting
0.0235 ±0.0004t
(4)
0.0237±0.002t
(5)
Arrested
Contracting
0.0375±0.0014
(7)
0.0386±0.0010
(10)
0.0400±0.0013
(10)
0.0444±0.0040
(8)
0.0541+0.0019t
(11)
0.0638±0.0016t
(10)
Cells were maintained for 2 days in medium to which 50 mM KC1 was added. Contraction was initiated by incubation in medium
containing 4 mM KC1 (day 0). Values are the mean±SEM, and the number of dishes are indicated by the figure in parenthesis.
*p<0.05 as compared with time 0.
tp<0.05 as compared with K, in arrested cells for same period of culture.
McDermott and Morgan
30
10
<
"
3. Ce// growth and total protein synthesis in KCl-arrested myocytes. Myocytes from
a single preparation were maintained as in Figure I. Values for total cell proteinlDNA (•) and
total protein synthesis (O) are the mean of
triplicate dishes. The amount of DNA over the
course of the experiment averaged 38.4 ±0.6 fig
(SEM) per culture dish and did not change
significantly with time in this preparation of
cells.
FIGURE
Q
o
0.
547
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Protein Synthesis in Cultured Heart Cells
20
_l
O
10
2
3
4
5
6
TIME IN CULTURE (DAYS)
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2 and 3 days of contraction, the apparent fractional
rate of protein synthesis increased by 30% and 24%
as compared to KCl-arrested cells. The magnitude
of the increase in protein synthesis was similar to
the increase in protein/DNA ratios at each time
period (Table 2).
The fractional rate of protein degradation (K,,)
was determined23 by the formula K g =K s -K d and
was used to compare the rates of protein degradation in contracting and KCl-arrested cells. Kg, the
fractional rate of protein accumulation, was determined from the formula P(t)=P(0)eK*<t) using linear
regression analysis of the protein/DNA ratios in
Table 2. The Kd values for cells after 2 and 3 days
of contraction were 0.0204/hr and 0.0189/hr respectively, which averaged 9.5% faster than the rate of
protein degradation in KCl-arrested cells at the
corresponding time points (0.0182/hr and 0.0177/hr).
The fractional rate of protein synthesis exceeded
degradation by an average of 25% in contracting
cells. In contrast, the average difference between
K, and Kd during KCl-arrest was 8%, which
reflected the slower rate of growth in these cells.
The relatively small differences in the degradation
rates between contracting and arrested cells suggested that the primary mode through which contraction facilitated accumulation of cell protein was
acceleration of the rate of protein synthesis.
Although greater ribosome content contributed to
growth of contracting myocytes, more efficient use
of existing components of the protein synthetic
pathway may also have contributed to the faster
rate. Efficiency of protein synthesis refers to the
rate of peptide chain initiation and elongation and is
affected by the availability of translational factors
and mRNA. The efficiency of protein synthesis can
be assessed by determining the percentage of total
RNA present as ribosomal subunits. When postmitochondrial supernatants were subjected to sucrose
gradient density centrifugation to resolve 40S and
60S subunits, approximately 18.8±2.9% and
21.8±3.8% of the total RNA applied to sucrose
gradients was present as subunit RNA in both KClarrested and contracting cells, respectively (three
preparations), after 2 and 3 days of contractile activity. Therefore, contraction did not appear to have an
effect on the efficiency of protein synthesis.
Modulation of RNA Content in Contracting Cells
RNA content, as expressed by RNA/DNA ratios
(Figure 1), was relatively constant during KC1 arrest.
This observation was confirmed by the data shown
in Table 2. In these experiments, however, the
RNA/DNA ratio was not sustained in KCl-arrested
cells, and an 8% decrease in the RNA/DNA ratio
occurred between days 3 and 6 of culture. The RNA
contents were analyzed by linear regression and
used to determine K, by dividing the slope (/xg
RNA/^ig DNA/hr) by the RNA/DNA ratios corresponding to each time point. Kg between 3 and 6
days of KC1 arrest was -0.0009/hr, which resulted
in a net loss in RNA content of 2.2%/day. In
contracting cells, there was a sustained increase in
the RNA/DNA ratio that was apparent on the first
day after the onset of contractile activity. The
accumulation of RNA in contracting cells was
reflected by values for Kg that averaged 0.0048/hr
over 3 days. An increase in total RNA per cell
reflected increased ribosome content because at
least 85% of total RNA was ribosomal.34 Thus, in
contracting cells, an increased ribosome content
paralleled accelerated rates of protein synthesis and
cell growth.
Effects of Contraction on RNA Synthesis
Rates of total RNA synthesis were measured to
assess the contribution of RNA synthesis to
increased RNA content in contracting cells. RNA
synthesis rates were calculated by measuring incorporation of [3H]uridine into [3H]UMP of RNA and
the specific radioactivity of intracellular [3H]UTP.
The primary source for UTP synthesis in cardiac
548
Circulation Research Vol 64, No 3, March 1989
FIGURE 4. Measurement ofRNA synthesis with
I3 H] uridine. Cells were labeled with 50 fiM
[3H]uridine (25 fiCilml). Specific radioactivities
of medium uridine and cellular UTP and incorporation of radioactivity into RNA were measured as described in ' 'Materials and Methods.''
Data are from a single cell preparation, but
similar results were obtained in three other
preparations.
INCUBATION TIME (HOURS)
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tissue is the salvage pathway which serves to reutilize UMP derived from RNA degradation.33 The
contribution of this pathway to UTP synthesis is
substantial because 1) the pool of UMP in RNA is at
least threefold greater than the cellular UTP pool36
and 2) the enzymes for de novo synthesis of uridine
are relatively inactive in cardiac muscle.33 A concentration of [3H]uridine in the medium (50 /AM)
that was 25 times greater than the usual plasma
concentration37 was used to expand the pool of
UTP and to maintain a constant specific activity of
uridine during 6 hours of labeling (Figure 4). During
the first hour of incubation with [3H]uridine, UTP
specific radioactivity rose rapidly presumably due
to expansion of the UTP pool as occurs in the
perfused rat heart and in HeLa cells. 3839 After 1
hour, UTP specific radioactivity was relatively stable during the remaining 5 hours of labeling, reaching approximately 70% of the specific radioactivity
of uridine in the culture medium. Even though
equilibration between uridine in the medium and
UTP was not observed after 6 hours, the incorporation of radioactivity into RNA was linear (Figure
4). Lack of equilibration of these specific radioactivities could have been due to slow turnover of a
pool of uridine nucleotide derived most likely from
RNA or to inhibition of uridine kinase by UTP. 35
From the point of view of measuring RNA synthesis, the expanded UTP pool was assumed to be
well-mixed as to serve as the precursor for RNA
synthesis in arrested and contracting cells. This
pool had a relatively stable specific activity between
30 minutes and 6 hours. Furthermore, the specific
activities of UTP at 6 hours were the same in
contracting and arrested cells (96±5%, n=12). UTP
specific radioactivities were determined routinely at
6 hours to correct for any variations in the precursor pool labeling in individual cell preparations and
was used to calculate the rate of RNA synthesis.
An increase in RNA content in contracting cells
could have resulted from a faster rate of RNA
synthesis, a decreased rate of RNA degradation or
some combination of changes in both rates. A
steady rate of RNA synthesis was observed in
KCl-arrested myocytes by day 3 in culture (Figure
5) and was associated with a relatively stable RNA
content. These findings indicated that from day 3
onward rates of RNA synthesis and degradation
were similar in arrested cells. Assuming that a wellmixed UTP pool served as the precursor in contracting and arrested cells, synchronous contractile
activity resulted in a progressively faster rate of
RNA synthesis that was significantly increased as
compared with noncontracting cells after 48 and 72
hours. The rate of RNA synthesis after 3 days of
contraction was similar to the rate observed upon
initiation of KG arrest on day 1 in culture.
The fractional rates of RNA synthesis and degradation were calculated to compare their relative
contributions to changes in RNA content in both
contracting and KCl-arrested cells. The rates of
RNA synthesis in Figure 5 were analyzed by linear
regression. The fractional rates of RNA synthesis
per hour (KJ were calculated from the corresponding synthesis rates extrapolated from the curve,
50
to 7
st
30
5a
i '
P I
20
10
TIME IN CULTURE (DAYS)
FIGURE 5. RNA synthesis in KCl-arrested and contracting myocytes. Cells were incubated as described in ' 'Materials and Methods" in serum-free medium to which 50
mM KCl was added in day 1. On day 3, cells were either
maintained in this medium (•) or allowed to contract by
incubation in medium containing 4 mM KCl (O). Values
are the mean±SEM for at least seven culture dishes.
'p<0.00l as determined by the Student's t test.
McDermott and Morgan
so
Protein Synthesis in Cultured Heart Ceils
549
-i 1000
to
Q
i
V)
40
-
900
30
-
800
<
z
20
-
700
o
10
UJ
0C
Z)
111
o
It
o
IT
a.
tr
111
-
600
o
o
FIGURE 6. The effects of adrenergic agonist and
antagonist on RNA content and total cell protein.
Cells were maintained in serum-free medium to
which 50 mM KCl was added until day 3, whereupon the indicated modifications were made. After
72 hours, RNA (open bars) and total protein (shaded
bars) per culture dish were measured simultaneously. NE, norepinephrine. Values represent the
mean±SEM of five culture dishes. Significance of
differences was assessed by AN OVA followed by
the Student-Newman-Keuls test for differences
between individual means. 'p<0.01 versus 50 mM
KCl-arrested myocytes.
KCL-mM
ADOITION(S)
5 urn PROPRANOLOL
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PHENTOLAMINE
assuming a value of 640 pmol UMP//xg ribosomal
RNA40 (K5=[pmol UMP/^tg RNA/hr]n-[640 pmol
VMP/fig RNA] (Table 2). The fractional rates of
RNA degradation (K^hr) were calculated by the
formula K g =K,-K d where Kg represents the fractional rate of RNA accumulation. The small decline
of the RNA/DNA ratios in KCl-arrested cells was
reflected by a Kd (0.0409/hr) that exceeded K,
(0.0400/hr) after 6 days in culture. By comparison,
contraction accelerated K,, and it exceeded Kd,
which was accelerated concomitantly. After 3 days
of contraction, K, was accelerated by 80% and
exceeded Kd (0.0592/hr) by 8%. The Kg accounted
for an increase of 0.35 jig RNA//ig DNA over 3
days of contraction, which represented a net increase
in RNA content of 46%. Since Kd was accelerated
by contraction, there was no additional increase in
RNA content after 2 days. Thus, an accelerated rate
of RNA synthesis accounted for a net accumulation
of RNA in contracting cells, but the potential
increase in RNA content was not achieved because
of simultaneous acceleration of RNA degradation.
When synchronously contracting cells were compared with KCl-arrested cells, the assumption was
made that the effect of contractile activity on cell
growth is mediated through direct stimulation of the
pathways of RNA and protein synthesis. The effects
of contractile activity were also examined by arresting myocytes on day 1 with 2x 10"5 M verapamil, a
calcium channel antagonist which lowered the contraction frequency of neonatal rat myocytes through
suppression of Ca2+ and Na+ influx.41 The rate of
RNA synthesis in verapamil-treated cells was 23.7
and 21.6 pmol UMP/^ig RNA/hr when measured on
days 5 and 6 during verapamil arrest. These rates
were similar to values obtained in KCl-arrested
cells on these days of culture (Figure 5). RNA
content and cell growth were compared in an experiment in which cells were arrested by the addition of
either 50 mM KCl or 1 x 10"5 M verapamil. After 6
days of arrest, RNA content as measured by RNA/
DNA ratios was 0.50+0.03 (/i=3) in KCl-arrested
cells compared with O.54±O.O3 for verapamil arrest.
Similarly, the protein/DNA ratio was 15.6±1.1 and
17.1 ±0.9 for KCl and verapamil arrest respectively.
Cells in which contractile activity was initiated by
removal of verapamil on day 3 had increased RNA/
DNA (0.69±0.08) and protein/DNA (22.7±0.07)
ratios. These ratios were increased to a similar
extent as produced by the onset of contraction by
reduction of the KCl concentration from 50 mM to
4 mM; 0.65±0.03 for RNA/DNA and 20.0±0.09 for
protein/DNA.
Effects of Norepinephrine on Cell Growth
Cessation of cell growth during KCl arrest was
attributed to a loss of contractile activity. The
ability of KCl-arrested myocytes to respond to a
growth stimulus other than contraction was tested
by addition of norepinephrine. Norepinephrine stimulated growth of sparsely plated neonatal myocytes
in culture.2 Myocytes were maintained in medium
containing 50 mM KCl until day 3, at which time 2
/xM norepinephrine was added (Figure 6). After an
additional 3 days of KCl arrest (a total of 6 days in
culture), norepinephrine treatment resulted in significantly more RNA/culture dish compared with
KCl-arrested cells incubated in the absence of the
hormone. Similarly, total protein increased in
norepinephrine-treated as compared to untreated
KCl-arrested myocytes. In contrast to studies in
which adrenergic activation of contractile activity
did not stimulate myocyte growth,10-12 the onset of
spontaneous contraction upon incubation in medium
containing 4 mM KCl resulted in proportional
increases in cell protein and RNA. The effects of
spontaneous contraction on cell protein and RNA
accumulation were not blocked by the adrenergic
antagonists propranolol and phentolamine.
550
Circulation Research Vol 64, No 3, March 1989
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Discussion
Neonatal rat myocytes in culture maintain many
of the differentiated properties of cardiac muscle
cells, including the ability to contract synchronously. The rate of contractile activity in culture is
established by groupings of pacemaker cells which
regulate contraction through a functional syncytial
network.42 Thus, culture densities that permit extensive patterns of intercellular contacts between individual pacemaker centers demonstrate regular rates
of synchronous contraction. In contrast, sparsely
plated cardiac muscle cells have less extensive
intercellular communication and contract irregularly when maintained in serum-free medium.4 Cells
cultured at sparse densities require serum or catecholamines to stimulate growth while synchronously contracting myocytes demonstrate hypertrophic growth in the absence of other stimuli.12-29
More recent work by Marino et al13 demonstrated
that neonatal rat myocytes maintained in serumfree media grew when contractile activity was stimulated by culture in low [Ko] medium. These findings suggest that synchronous contraction at constant
rates may be involved in regulating cell growth.
Noncontracting myocytes were used as a model
to study the effects of spontaneous, synchronous
contraction on cell growth for two primary reasons.
First, RNA content and rates of RNA and protein
synthesis stabilized during KC1 arrest. The limited
rate of growth as measured by the protein/DNA
ratio provided an improved opportunity to detect
effects of contraction. The early phase of growth
that occurred in noncontracting cells may have
been attributable to cellular repair.43 Thus, the
effects of a growth stimulus could be more easily
observed by comparison to nongrowing cells after 2
days of KG arrest. Secondly, contractile activity
was easily initiated by returning to 4 mM KG in the
medium, providing the opportunity to study early
and late effects of contraction on cell growth and
macromolecular synthesis in defined media containing physiological [K + ] o . Resumption of synchronous contraction stimulated cell growth and the rate
of protein synthesis in parallel. RNA content
increased concomitantly. The rates of protein degradation were not affected significantly by onset of
contractile activity. These findings indicated that
contraction stimulated growth by increasing the
capacity for protein synthesis. Similarly, RNA content increased during myocyte hypertrophy in vitro
in response to norepinephrine.7 Increased RNA
content during compensatory cardiac growth was
observed earlier in response to pressure overload44
thyrotoxicosis15 and during postnatal left ventricular development.45 Thus, increased production of
ribosomes is an important component of cell growth
both in vivo and in vitro.
Measurements of protein synthesis rates were
performed by two methods. First, protein synthesis
rates were calculated after pulse-labeling myocytes
with 0.4 mM [3H]PHE and dividing the rate of
incorporation of [3H]PHE into total cell protein by
the specific radioactivity of [3H]PHE tRNA, the
immediate precursor for protein synthesis. Despite
the fact that the extracellular and intracellular pools
of [3H]PHE equilibrated, the specific radioactivity
of PHE-tRNA was approximately 56% of extracellular PHE. Other studies employing cultured cells
have shown that the pool of radiolabeled aminoacyltRNA did not attain a specific radioactivity equivalent to the pool of radiolabeled amino acid in the
culture medium.313246 The relationship between the
extracellular, intracellular, and aminoacyl-tRNA
pools are difficult to predict during pulse labeling
because conflicting findings have been reported. In
agreement with our results, the specific radioactivity of intracellular tyrosine equilibrated with the
extracellular pool during pulse labeling of neonatal
rat myocytes in culture.10 However, in studies
utilizing embryonic chick myocytes, intracellular
leucine specific radioactivity did not equilibrate
with the extracellular pool and was lower than the
pool of leucyl-tRNA.32 These observations, taken
together with our results, suggested that phenylalanyl-tRNA was synthesized from the extracellular
or intracellular PHE pools or both along with PHE
derived from protein degradation. Preferential reutilization of unlabeled PHE from protein degradation was not observed during pulse labeling with 0.4
mM PHE in the perfused heart of adult rats.30 The
lack of equilibration of the specific radioactivities of
medium, intracellular, and tRNA-bound phenylalanine in cultured myocytes could reflect metabolic
changes resulting from the transition to tissue culture, the use of neonatal cells, and the lack of serum
in the incubation medium. Frelin47 found that addition of serum decreased the rate of protein degradation in neonatal rat myocytes.
These results underscored the importance of determining the specific radioactivity of aminoacyltRNA for measurements of protein synthesis rates
by pulse labeling in cultured cells. We confirmed
these measurements of protein synthesis rates by a
second method, dual isotope labeling. The dual
isotope method had the advantage over conventional pulse labeling because direct measurement of
the phenylalanyl-tRNA pool was not required to
measure fractional rates of protein synthesis. The
values for K,, as measured at days 5 and 6 during
KC1 arrest, were identical to the corresponding
fractional rates calculated from pulse-labeling experiments (Figure 3).
Protein synthesis rates in contracting and KC1arrested myocytes were compared by the dual isotope method. Measurements of K, require a steady
state such that the rates of protein synthesis and
degradation are equal.48 Noncontracting myocytes
were in a steady state by day 3 in culture and were
pulse-labeled for 4 hours on days 5 and 6 during
KC1 arrest to obtain values for K,. In contrast,
measurements for Ks in contracting cells were
McDermott and Morgan
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apparent rates because a steady state was not
established. If the fractional rate of protein turnover
was assumed to be constant over the duration of
pulse labeling with [3H]PHE in contracting cells, Kd
was 9.5% faster than in arrested cells. Further
studies will be required to directly measure rates of
protein degradation during concentration stimulated growth.
Since the ability of contraction to stimulate myocyte growth was assessed by comparison to KCI
arrested cells, it was critical to establish that the
cessation of growth was a direct result of diminished contractile activity during KC1 arrest. The
following lines of evidence suggest that 50 mM
KCl-arrested cells were a suitable model with which
to compare cell growth during contraction. 1) Cell
growth occurred during the first 2 days of KC1
arrest. 2) Rates of protein and RNA synthesis were
stable between days 3 and 6 of arrest. 3) Synchronous contractile activity resumed within one hour
after removal of 50 mM KCI which suggested that
arrested cells maintained the functions of the
myofibrils,3 sarcolemma, and sarcoplasmic reticulum. 4) The balance between rates of peptide chain
initiation and elongation was not affected by 50 mM
KCI because the percentage of RNA in ribosomal
subunits remained low (approximately 20%) and did
not differ in comparison to contracting cells. 5) Cell
growth and RNA content increased in response to
the continued presence of norepinephrine in KClarrested cells (Figure 6). Because KCl-arrested cells
maintained their ability to respond to restoration of
a normal extracellular potassium concentration with
resumption of growth and beating and to addition of
an adrenergic agonist with increased RNA and
protein content, KCl-arrested cells were a satisfactory control for changes in RNA and protein metabolism during growth.
Although contraction is discussed in this article
as the stimulus for growth, the parameter that most
closely links the process of contraction to a hypertrophic response is unknown. It has been proposed
that passive stretch of the ventricular wall after
imposition of elevated aortic pressure is responsible
for stimulating growth in intact heart tissue.18 Stretch
of adherent cells as would occur during repetitive
contraction and expansion against the resistance of
intracellular components and the fluid medium may
occur in confluent myocyte cultures. A relation
between cellular morphology and growth has been
observed in cultured adult feline cardiocytes and is
dependent upon the ability of the cells to adhere to
the substrate and establish a resting diastolic length.49
Initiation of contractile activity by incubation in low
[K + ] o medium resulted in a relatively higher content
of RNA as compared to quiescent adherent cells.
Furthermore, an increase in the diastolic resting
length by 10% on a deformable substrate elevated
RNA content in these cells.10 If mechanical deformation of the myocyte is not directly linked to
growth, changes in membrane potential and intra-
Protein Synthesis in Cultured Heart Cells
551
cellular concentrations of Ca2+, Na + , or Cl could
be of importance for the direct control of RNA and
protein synthesis in contracting cells.
Inhibition of contractile activity by KCI arrest
has been investigated using embryonic chick
myotubes.51-52 Protein synthesis was unaffected by
incubation in medium containing 12 mM KCI, but
turnover of myofibrillar proteins was accelerated
between 5 and 6 days of KCI arrest. Although
turnover of contractile proteins was not investigated in the present experiments, neonatal cardiac
myocytes accumulate myosin and resume contractile activity during the first 4 days of KCI arrest.28
Stable rates of protein synthesis were obtained in
arrested cardiac myocytes (Figure 3). When spontaneous contraction of skeletal myotubes resumed
in 4 mM KCI, reexpression of the embryonic myosin heavy chain occurred.52 Contraction may be a
common signal affecting protein metabolism in both
skeletal and cardiac muscle.
Faster cell growth in contracting as compared
with noncontracting neonatal rat myocytes is mediated through an increased capacity for protein synthesis, as assessed by total RNA content. Our
primary interest in this model is to study regulation
of ribosome formation and coordination of synthesis of various ribosomal components. Ribosome
formation requires the transcription of ribosomal
DNA and the processing of the gene product into
18S, 28S, and 5.8S rRNA." Assembly of rRNA into
mature 40S and 60S subunits requires approximately 70 ribosomal proteins.54 Availability of these
proteins may be limiting for ribosome production
through controls of their formation at the transcriptional, posttranscriptional, or translational levels. A
thorough study of" ribosome formation in heart
muscle is complicated by the slow turnover of
ribosomes55 and the modest extent to which ribosome formation is accelerated during hypertrophy
in vivo. l517 In the present model of myocyte growth,
rates of ribosomal RNA and ribosomal protein
synthesis can be measured over either short or
prolonged labeling periods. In the latter circumstance, a modest acceleration of the rate of ribosome formation will be amplified by acting for
longer times. Secondly, because arrested cells have
reduced rates of RNA and protein synthesis, the
effect of contraction is measured against a lower
baseline, and, as a result, the magnitude of the
effect is enhanced.
Acknowledgment
The authors thank Bonnie Kleinhans, Kathryn
Giger, and John Price for their excellent technical
assistance and Donna Morgan for typing this manuscript.
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KEY WORDS
cell culture
contraction • protein synthesis • myocyte
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Contraction modulates the capacity for protein synthesis during growth of neonatal heart
cells in culture.
P J McDermott and H E Morgan
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 1989;64:542-553
doi: 10.1161/01.RES.64.3.542
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