Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Health Science Center, Houston, Texas 77030
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ABSTRACT |
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The purpose of
this study was to determine whether mechanical stretch or serum
availability alters pretranslational regulation of skeletal -actin
(SkA) in cultured striated muscle cells. Chicken primary skeletal
myoblasts and cardiac myocytes were plated on collagenized Silastic
membranes adherent to nylon supports and stretched 8-20% of
initial length 96 h postplating. Serum dependence of SkA gene
regulation was determined by maintaining differentiated muscle cells in
growth/differentiation (G/D; skeletal myotubes, 10% horse serum-2%
chick embryo extract; cardiac myocytes, 10% horse serum) or
growth-limiting (G-L; 0.5% horse serum) medium. Skeletal myotubes had
higher SkA mRNA and SkA promoter activity in G/D than in G-L medium.
Cardiac myocyte SkA mRNA was higher in G-L than in G/D medium. Serum
response factor (SRF) protein binding to serum response element 1 (SRE1) of SkA promoter increased in skeletal cultures in G/D compared
with G-L medium. Western blot analysis demonstrated that increased
SRF-SRE1 binding was due, in part, to increased SRF protein. Stretching
skeletal myotubes in G-L medium reduced SkA mRNA and repressed SkA
promoter activity. The first 100 bp of SkA promoter were sufficient for
stretch-induced repression of SkA promoter activity, and an intact
transcriptional enhancer factor 1 (TEF-1) binding site was necessary
for this response. Serum and stretch appear to repress SkA promoter
activity in skeletal myotubes through different DNA binding elements,
the SRE1 and TEF-1 sites, respectively. Stretching increased SkA mRNA in cardiac myocytes in G-L medium but did not alter SkA mRNA level in
cardiac cells in G/D medium. These results demonstrate that stretch and
serum interact differently to alter SkA expression in cultured cardiac
and skeletal muscle cells.
skeletal myotubes; cardiomyocytes; hypertrophy; serum response factor; transcriptional enhancer factor 1
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INTRODUCTION |
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ENVIRONMENTAL STIMULI CAN induce alterations of cell morphology and gene expression (26). Mechanical stretch is an example of a stimulus that induces striated muscle cell hypertrophy in the animal (31) and in culture (25, 35). Gene expression during stretch-induced cardiac myocyte hypertrophy has been extensively studied (26); however, considerably less is understood about the regulation of gene expression in stretched skeletal myotubes. A well-documented pattern of developmental gene reexpression occurs during cardiac myocyte hypertrophy and is considered a marker of cardiac enlargement (27). A similar sequence of gene expression has not been clearly defined during the hypertrophy of cultured skeletal myotubes. Although skeletal and cardiac muscle are both classified as striated muscle, several well-characterized differences exist in their biological and functional properties. These differences include the degree of contractile activity, regenerative capacity after injury, number of nuclei, stem cell availability, and myogenic transcription factor abundance. Thus the molecular mechanisms regulating cardiac myocyte gene regulation during stretch-induced hypertrophy may not be similar to the molecular mechanisms signaling skeletal myotube hypertrophy.
The study of skeletal -actin gene regulation provides an excellent
paradigm for examining stretch-induced gene regulation in cardiac and
skeletal myocytes because of its differential gene regulation in
development and postnatal life. Skeletal
-actin is a primary
component of skeletal muscle thin filament and is expressed after
myoblast differentiation. Skeletal
-actin mRNA levels and promoter
activity are altered during stretch-induced hypertrophy of skeletal
muscle in vivo (2, 3). Stretch overloading the chicken anterior
latissimus dorsi (ALD) muscle increases skeletal
-actin mRNA between
the third and sixth day of stretch (3), and skeletal
-actin promoter
activity is induced at 6 days of stretch (2). In cardiac muscle,
skeletal
-actin is an isoform that is downregulated during
development, and its expression level is dependent on both the species
and age of the animal (28). During cardiac hypertrophy, skeletal
-actin gene expression is induced both in the animal (28) and in
culture (27). Skeletal
-actin induction in cardiac myocytes is part
of a reexpression of developmental genes that are used as markers of
cardiac hypertrophy. Thus the stretch-induced signals that trigger an
actin isoform switch in cardiac myocytes may not be similar to the
regulation modulating skeletal
-actin expression levels in stretched
skeletal myotubes.
Myocyte cultures are supplemented with serum containing an abundance of
proteins, polypeptide growth factors, hormones, nutrients, metabolites,
and minerals (10). Culture medium is supplemented to facilitate overall
cell viability and growth. Manipulating the concentration of serum in
the culture medium is a method for switching cells into a proliferating
or differentiating state (10). Skeletal myoblast cultures are sensitive
to alterations in the serum concentration of the medium, and switching
from a high serum concentration to a low serum concentration induces proliferating myoblasts to differentiate, fusing to form myotubes. After myoblast differentiation, the serum concentration of the culture
medium still affects cellular regulation. Serum withdrawal increases
protein degradation in differentiated skeletal myotubes (36). Adding
serum back to serum-starved myotubes cultures increases myotube protein
synthesis (12). Supplementing differentiated myotubes after serum
withdrawal with insulin or insulin-like growth factor I (IGF-I) induces
an increase in cell number, as well as an increase in mean myofiber
cross-sectional area (34). Skeletal myotube cultures subjected to
static stretch increase amino acid incorporation into cellular protein,
increase accumulation of total protein, and increase the accumulation
of myosin heavy chain (MHC) protein, and these responses were similar
for a range of static stretch varying from 7.5 to 13% of resting
length (35). Varying the serum levels in primary chick myotube cultures
has been used to demonstrate that the stretch-induced myotube growth process contains both serum-independent and serum-dependent processes (33). The serum dependence of skeletal -actin gene regulation in
differentiated skeletal myotubes has not been investigated. Furthermore, the interaction of serum and passive stretch on skeletal
-actin gene regulation in skeletal myotube and cardiac myocyte cultures has not been determined.
It is not known whether stretch induces skeletal -actin mRNA
expression in skeletal myotubes or whether there is any stretch-induced regulation at the level of the skeletal
-actin promoter. Stretching cardiac myocytes cultured in serum-free medium induces
c-fos and skeletal
-actin mRNA
concentrations, and the stretch responsiveness of
c-fos is regulated at the level of the
promoter (25, 27). Skeletal
-actin promoter regulation has not been
investigated in stretched cardiac myocytes. Skeletal
-actin promoter
regulation has been examined in cardiac myocytes induced to hypertrophy
by transforming growth factor-
(TGF-
) and
-adrenergic
stimulation, and the first 100 bp of the actin promoter are sufficient
for induction with these stimuli (16, 19). It is not certain whether mechanical stretch signaling works through a similar type of
regulation. The primary purpose of this study was to examine whether
serum availability, mechanical stretch, and any interaction between these two variables alter the pretranslational regulation of skeletal
-actin in cultured striated muscle cells. A second purpose of the
study was to compare the stretch responsiveness of skeletal
-actin
expression in two types of striated muscle lineages, skeletal and
cardiac myocytes.
We demonstrate that serum availability and stretch regulate skeletal
-actin pretranslationally in skeletal myotubes and cardiac myocytes.
In skeletal myotubes, serum availability and stretch use different
regulatory mechanisms to decrease skeletal
-actin promoter activity.
Serum alters serum response factor (SRF)-serum response element 1 (SRE1) binding interactions, whereas mechanical stretch needs an intact
transcriptional enhancer factor 1 (TEF-1) element to mediate a decrease
in promoter activity. The effect of stretch on skeletal
-actin mRNA
concentration of cardiac myocytes is altered by the serum content of
the culture medium; skeletal
-actin mRNA is increased during stretch
in low-serum conditions. These results demonstrate that skeletal
-actin gene expression is responsive to both serum availability and
stretch in cardiac myocytes and skeletal myotubes; however, serum
availability and stretch alter the pretranslational regulation of
skeletal
-actin differently in these two types of striated muscle.
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MATERIALS AND METHODS |
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Primary embryonic myoblast culture. White Leghorn eggs from the Texas A&M Poultry Science Dept. were incubated at 38°C. Primary myoblast cultures were established by mechanically dissociating breast tissue, dissected free of skin and connective tissue, from 11-day-old chicken embryos as described previously (13). Briefly, myoblasts were preplated two times in plastic 100-ml culture flasks, and cell concentration was determined. Myoblasts were then plated on rat collagen-coated Silastic membranes (Specialty Manufacturing, Saginaw, MI) adherent to nylon support devices (30) at a density of 900-960 cells/mm2 in 15 ml of plating medium and placed in a humidified 5.0% CO2 incubator at 37°. Cells were localized over the Silastic membrane by a 23-mm-diameter ring. Plating medium consisted of MEM (Sigma) supplemented with 50 U/ml gentamicin, 10% heat-inactivated horse serum (HIHS), and 5% chick embryo extract (CEE).
Primary embryonic cardiac myocytes cultures were established by enzymatic digestion of the 11-day-old chicken embryo ventricles. The cardiac myocyte population was further purified by centrifugation through a Percoll gradient as described previously (19). Purified cardiac myocytes were then plated on rat collagen-coated Silastic membranes (Specialty Manufacturing) adherent to nylon support devices (30) at a density of 1,200-1,800 cells/mm2 in 15 ml of plating medium and placed in a humidified 5.0% CO2 incubator at 37°. Cells were localized over the Silastic membrane by a 23-mm-diameter ring. Plating medium consisted of DMEM (Sigma) supplemented with 50 U/ml gentamicin and 10% HIHS. Approximately 30-36 h postplating or posttransfection (see below), the skeletal myoblast plating medium was replaced with growth/differentiation (G/D) medium, consisting of MEM supplemented with 50 U/ml gentamicin, 10% HIHS, and 2% CEE. After ~24-30 h in G/D medium, one-half of the skeletal myotube cultures were randomly selected, washed with Hanks' balanced salt solution (HBSS, Sigma), and placed in growth-limiting (G-L) medium consisting of MEM supplemented with 50 U/ml gentamicin and 0.5% HIHS. After ~12-24 h in G-L medium, cells were subjected to the cell stretch protocol (see Cell stretch).Transient transfection of myocytes.
Skeletal myoblast or cardiac myocyte cultures were transfected
24-36 h postplating using Lipofectamine reagent (GIBCO). The skeletal -actin promoter constructs have been previously described (2, 3). The mutations to the SRE1 (M15) and TEF-1 (M11) elements of the
99-bp skeletal
-actin promoter have been previously described
(2). Two micrograms of reporter plasmid were cotransfected with one
microgram of a plasmid containing the cytomegalovirus (CMV) promoter
directing the expression of galactosidase (CMV/GAL). The CMV/GAL
construct was used to correct for variations in transfection efficiency. DNA was mixed with the Lipofectamine reagent in DMEM for
15-30 min. Cell stretchers were washed two times with HBSS and
then placed in DMEM without antibiotic. The transfection reaction was
then added to the cells and kept in place for 3-5 h in the culture
incubator. On completion of transfection, the transfection medium was
replaced. Skeletal cultures were placed in G/D medium consisting of MEM
supplemented with gentamicin, 10% HIHS, and 2% CEE. Cardiac myocyte
cultures were placed in G/D medium consisting of DMEM supplemented with
gentamicin and 10% HIHS. Because most studies producing hypertrophy of
cardiac myocytes in culture use no serum in the culture medium, we
employed a G-L medium of 0.5% horse serum for comparisons to the literature.
Cell stretch. Skeletal myotubes or cardiac myocytes were subjected to static stretch 80-90 h postplating. Cells were stretched 8-24% of resting length by the turning of the support bolts on the stretcher apparatus. One turn of the support bolts was consistent with a 3.3% lengthening of the membrane, when preset to a 24-mm length. Cell stretch was performed by holding the support above the culture dish with sterile instruments and turning the bolts a specific number of rotations with sterile instruments. The entire stretch procedure lasted ~20-45 s. Previous data by Vandenburgh and Kaufman (35, 36) established that cells are stretched by these procedures. They observed a linear increase in the longitudinal length of myotubes with progressive increases in stretch of membranes from 5 to 20% on a stretching frame (35). Vandenburgh and Kaufman (35) found that a continuous linear stretch of 7.5-13% gave results similar to stretching by 10.8%. Sadoshima et al. (27) observed that a longitudinal stretch of the silicone substrate by 20% resulted in a comparable (20 ± 1%) increase in cardiac myocyte cell length (or width) in the direction of stretch. They found no evidence of cardiac injury to myocytes from the stretch. The cells were then maintained in this condition for 6-24 h until harvesting.
mRNA analysis.
Total RNA was extracted using the TRIzol method (GIBCO). RNA
concentration and purity were determined by ultraviolet
spectrophotometry. Northern blot analysis was performed as previously
described (2). Eight to ten micrograms of total RNA were fractionated
on a denaturing 1% agarose gel (1× MOPS and 6.7% formaldehyde)
and then transferred to a nylon membrane by capillary action. All
probes for Northern analysis were made by random priming as previously
reported (2). The chicken skeletal -actin probe has been described
previously (2, 7). Hybridization of the labeled probes with the RNA containing membrane was performed as previously described (2). Membranes were then visualized by autoradiography (
80°C,
3-40 h) and quantified by densitometry scanning (BioImage,
Millipore) to obtain an integrated optical density (IOD) that was used
to calculate IOD mRNA per microgram of total RNA. The
quantity of mRNA was normalized to an 18S rRNA probe, using
autoradiography and image analysis.
Total RNA determination. Total RNA was quantified using the method of Fleck and Munro (9).
Whole cell protein extracts.
Protein extracts were made from primary cultures as previously reported
(18). Cultures were washed two times in PBS and then scraped into 1 ml
of ice-cold PBS. Cells were centrifuged (15,000 rpm, 5 min, 4°C),
and the pellet was resuspended in 0.1 ml of extract buffer (10 mM
HEPES, pH 7.9, 0.5 M KCl, 0.5 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethylsulfonylfluoride, and 5% glycerol). Cells were lysed by
three freeze-thaw cycles and incubated on ice for 30 min with regular
vortexing. Extracts were then centrifuged (15,000 rpm, 10 min,
4°C), aliquoted, and stored at 80°C. The protein
concentration of whole cell extracts was determined by the
detergent-compatible (DC) Lowry assay (Bio-Rad). Whole
cell extracts were used for gel mobility shift assays and Western blots.
Mobility shift assay.
Gel mobility shift assays were performed in a manner similar to that
previously described (2). Five micrograms of whole cell protein extract
were used for binding reactions. SRF-SRE1 binding
complexes were further characterized by using hyperimmune serum made
against SRF (Ref. 7; gift from R. J. Schwartz, Baylor College of
Medicine, Houston, TX). Supershifted complexes were produced by adding
1 µl/reaction of SRF serum (2). The skeletal -actin SRE1
double-stranded oligonucleotide for mobility shift experiments
comprised nucleotides
100 to
73
(5'-GCCCGACACCCAAATATGGCGACGGCCG-3') of the skeletal
-actin promoter. The skeletal
-actin TEF-1 double-stranded oligonucleotide for mobility motif (nt
70 to
55) was
5'-AGCTTGCCGCATTCCTGGGGG-3' (19). All oligonucleotides were end labeled with
[32P]ATP using T4
polynucleotide kinase. The DNA binding assay was evaluated by
electrophoresis in a 5% polyacrylamide gel in 0.5× Tris-glycine buffer.
Western blot analysis. Western blot analysis was performed as previously reported (3). The protein concentration of whole cell extracts was determined by the DC Lowry assay (Bio-Rad). Fifty micrograms of protein were incubated in duplicate (15 min, 65°C) with an equal volume of protein sample buffer (20% glycerol, 6% SDS, 0.125 M Tris, pH 6.8, and 0.5% bromphenol blue) and fractionated on a 10% SDS-polyacrylamide gel (29:1 acrylamide-bisacrylamide, 150 V, 25°C, 4 h). One gel was then stained with Coomassie blue, and the second was electrophoretically transferred to a nitrocellulose membrane (300 mA, 4°C, 14 h). After transfer, the membrane was Ponceau S stained to verify transfer. The membrane was soaked in 1× TTBS (Tween-Tris-base-NaCl, pH 7.6) and then blocked in 5% milk-1.0% BSA for 1 h at 25°C. The SRF primary antibody (Santa Cruz) was added (1:500 dilution) to the membrane in 2.5% milk-1.0% BSA and incubated for 2 h at 25°C. The membrane was washed three times for 5 min each in TTBS and then incubated (1 h) with goat anti-rabbit secondary antibody conjugated with alkaline phosphatase. The secondary antibody was visualized by enhanced chemiluminescence (Amersham Life Sciences) as per manufacturer instructions and quantified by densitometry scanning (Bio Image, Millipore, Ann Arbor, MI).
Statistics. The data are expressed as means ± SE. Student's t-test was used to determine differences between the treatment and control cultures. The level of significance was set at P < 0.05.
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RESULTS |
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Skeletal -actin mRNA concentration in skeletal
myotubes.
The concentration of skeletal
-actin mRNA in skeletal myotubes was
altered by the concentration of serum in the culture medium. Skeletal
-actin mRNA concentration per microgram of total RNA increased 62%
(P = 0.0098) in skeletal myotubes
maintained in G/D medium compared with skeletal myotubes placed in G-L
medium (Fig.
1A).
18S rRNA concentration per microgram of total RNA was not different
(P = 0.99) between
skeletal myotubes maintained in either G-L medium (1.266 ± 0.180 IOD 18S rRNA; n = 8) or G/D medium
(1.262 ± 0.269 IOD 18S rRNA; n = 9).
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Skeletal -actin mRNA concentration in cardiac
myocytes.
Skeletal
-actin mRNA levels were also examined in cardiac myocytes.
Unlike the effect of serum increasing skeletal
-actin mRNA
concentration in skeletal myotubes, skeletal
-actin mRNA concentration decreased 32% (P = 0.027) in control cardiac myocyte cultures maintained in G/D medium
compared with cardiac cultures maintained in G-L medium (Fig.
3A).
Skeletal
-actin mRNA was previously shown to increase during stretch
of cultured myocytes (27). Our current study extends these findings by
reporting that skeletal
-actin mRNA expression in stretched cardiac
myocytes is dependent on the availability of serum for the cardiac
myocyte. Cardiac myocytes maintained in G-L medium increased skeletal
-actin mRNA concentration 67% (P = 0.0216) in stretched cultures compared with control cultures (Fig.
2C). Cardiac myocytes maintained in G/D medium demonstrated no significant difference (24%,
P = 0.36) between control and
stretched cultures (Fig. 3A).
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SRF mRNA in cardiac myocytes.
SRF mRNA concentration was examined in cardiac myocytes because SRF
mRNA increases during stretch-induced skeletal muscle hypertrophy in
the chicken (2). Additionally, the expression of SRF mRNA in stretched
cardiac myocytes has not been previously reported. The serum
concentration of the culture medium altered SRF mRNA concentration. SRF
mRNA concentration decreased 56% (P = 0.006) in cardiac myocytes maintained in G/D medium compared with
cardiac myocyte cultures maintained in G-L medium (Fig.
3B). Cardiac cultures maintained in
G/D medium increased the concentration of SRF mRNA 85%
(P = 0.019) in stretched cultures
compared with control cultures (Fig.
3B). Cardiac myocytes maintained in
G-L medium did not demonstrate a significant difference (40%,
P = 0.24) in SRF mRNA between control
and stretched cultures (Fig. 3B).
SRF mRNA was induced by stretch under conditions in which skeletal
-actin mRNA was not induced, and SRF mRNA was not significantly elevated by stretch under conditions in which skeletal
-actin mRNA
was increased. These data suggest that induction of SRF and skeletal
-actin mRNAs are not correlated during static stretch of cardiac myocytes.
Skeletal -actin promoter analysis.
Transient transfection assays were used to examine skeletal
-actin
promoter regulation during the stretch of striated muscle. The
424-bp skeletal
-actin promoter is sufficient for
tissue-specific and developmental regulation in cultured myocytes (1).
The activity of the
424 skeletal
-actin promoter generally
followed the mRNA expression pattern of skeletal
-actin. Serum
availability altered skeletal
-actin promoter activity in
differentiated skeletal myotubes. The
424 skeletal
-actin
promoter activity was increased 105%
(P = 0.00001) in skeletal myotubes
maintained in G/D medium compared with skeletal myotubes placed in G-L
medium (Fig.
4A). There was no stretch-induced difference in reporter activity from the
skeletal
-actin promoter between control and stretch cultures in G/D
medium for any stretch paradigm employed (data not shown). Stretch
decreased skeletal
-actin promoter activity in differentiated skeletal myotubes maintained in G-L medium. Promoter activity was
significantly decreased 24% (P = 0.004) in primary myotube cultures stretched 8% [394,482 ± 20,783 relative light units (RLU)/GAL; n = 9] in G-L medium,
relative to control cultures (522,838 ± 35,838 RLU/GAL;
n = 10). Stretching primary myotubes
16% of resting length decreased
424 skeletal
-actin promoter
activity 34% (P = 0.006) in stretched
cultures relative to control cultures (Fig. 4B). Deletions of the skeletal
-actin promoter revealed that the
99-bp and
77-bp
promoters were sufficient for this stretch-induced repression of
skeletal
-actin promoter activity (Fig.
4C). A skeletal
-actin promoter
construct with a mutated TEF-1 element (M11) demonstrated that a
functional TEF-1 element is necessary for stretch-induced repression of
the skeletal
-actin promoter (Fig.
4C). A mutation (M15) of SRE1
dramatically reduced promoter activity, which was previously reported
both in cell culture (17) and in the animal (2).
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Protein binding to SRE1 and TEF-1.
Protein-DNA interactions were analyzed during static stretch and
altered culture medium conditions. Gel mobility shift assays were
performed on whole cell protein extracts from skeletal myotubes using
oligonucleotides that corresponded to SRE1 and TEF-1 of the skeletal
-actin promoter. The SRE1 oligonucleotide formed binding complexes
with SRF and yin yang 1 (YY1) in both control and stretched extracts,
regardless of culture conditions (Fig. 6).
The ratio of SRF to YY1 binding to SRE1 was increased 57% (P = 0.00005) in cultures maintained
in G/D medium (0.33 ± 0.01 IOD/µg protein;
n = 4) compared with control cultures
maintained in G-L medium (0.21 ± 0.01 IOD/µg protein;
n = 4) (Fig. 6). This difference in SRF-to-YY1 ratio was due to increases in SRF binding rather than to alterations in YY1 binding to SRE1. The SRF-SRE1 binding
complex was greater in control cultures maintained in G/D medium (12.90 ± 0.25 IOD/µg protein; n = 4)
than in control cultures maintained in G-L medium (8.60 ± 0.47 IOD/µg protein; n = 4 IOD). There
was no difference in the YY1-SRE1 binding complex in control cultures
maintained in either G/D medium (39.40 ± 0.75 IOD/µg protein;
n = 4) or G-L medium (40.60 ± 1.40 IOD/µg protein; n = 4) (Fig. 6).
There was no alteration in binding at SRE1 with static stretch,
regardless of culture conditions (data not shown). These data suggest
that the increased actin promoter activity in skeletal myotube cultures
maintained in G/D medium, compared with control cultures in G-L medium,
may be due to increased SRF-SRE1 binding interactions; however, the
stretch-induced actin promoter repression in G-L medium does not appear
to work through the same mechanism (Fig.
4C).
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SRF protein in skeletal myotube cultures. Western blot analysis of whole cell protein extracts from skeletal myotube cultures were analyzed for SRF protein content (Fig. 8). There was no difference in SRF protein concentration with static stretch, regardless of culture conditions. SRF protein concentration did not demonstrate a statistical difference between control and stretched cultures in either G/D (2,444 ± 615 vs. 2,675 ± 173 SRF IOD/actin IOD, respectively; n = 3) or G-L medium (1,697 ± 314 vs. 1,309 ± 279 SRF IOD/actin IOD, respectively; n = 4). The concentration of SRF protein was increased 70% (P = 0.010) in pooled control and stretched skeletal myotube cultures maintained in G/D medium (2,560 ± 290 SRF IOD/actin IOD; n = 6) compared with pooled control and stretched skeletal myotube cultures maintained in G-L medium (1,503 ± 208 SRF IOD/actin IOD; n = 8). There was no significant difference in the concentration of Coomassie blue-stained actin protein with static stretch, regardless of culture conditions (data not shown). These data suggest that the decreased SRF protein binding to SRE1 in control cultures maintained in G-L medium is due, in part, to a decreased SRF protein concentration.
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DISCUSSION |
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This study demonstrates that the pretranslational regulation of
skeletal -actin in cultured striated myocytes is responsive to
mechanical stretch and that the exposure of the cell to serum modulates
the stretch response. The interaction of mechanical stretch with serum
availability altered skeletal
-actin regulation pretranslationally,
and this pretranslational regulation was demonstrated at the levels of
mRNA concentration and promoter activity. Skeletal
-actin regulation
was modulated by stretch and serum in both cardiac and skeletal
myocytes; however, the effect of the stretch-serum interaction was
different in these two types of striated muscle. Skeletal
-actin
mRNA concentration was significantly reduced in stretched skeletal
myotubes maintained in G-L medium, whereas there was an increase in the
concentration of skeletal
-actin mRNA in stretched cardiac myocytes
under the same conditions. There was no stretch effect on skeletal
-actin mRNA concentration when skeletal myotubes or cardiac myocytes
were cultured in optimal conditions for their cell growth and
viability. These data demonstrate that the stretch stimulus is not
sufficient to further alter skeletal
-actin mRNA concentration when
serum and growth factors are present in abundance.
The lack of skeletal -actin mRNA induction in stretched skeletal
myotubes might seem surprising, since static stretch has been shown to
induce protein accumulation in skeletal myotubes (35). However, at the
onset of stretch-induced hypertrophy of the chicken ALD muscle,
skeletal
-actin mRNA concentration is significantly reduced (2)
while actin protein synthesis rates are increased (11). These data
suggest that translational and/or posttranslational regulation
is important for the increase in actin protein synthesis at the onset
of stretch-induced hypertrophy in the living animal. Later in the time
course of stretch-induced muscle growth of chicken ALD, skeletal
-actin mRNA concentration increases to control levels (3) and
skeletal
-actin promoter activity is also increased (3). We were
unable to maintain myotube cultures for the duration necessary to test
long-term exposure to a stretch stimulus. Our current data demonstrate
that the availability of serum and growth factors can alter the
stretch-induced pretranslational regulation of skeletal
-actin in
skeletal myotubes. These facts, taken together with animal data, bring
forward the question of whether stretch is sufficient to induce
skeletal
-actin mRNA expression in skeletal muscle.
A mechanical stretch stimulus may require additional cofactors
and/or growth factor signaling to induce skeletal -actin
gene regulation in skeletal muscle. Stretch induction of growth factors has been demonstrated to follow varied time courses both in the animal
and in culture (8, 24). IGF-I mRNA is not induced in the stretched
chicken patagialis muscle until after the second day of stretch
overload (8). Components of stretch-induced growth in skeletal myotubes
demonstrate both serum-dependent and serum-independent regulation (33).
Possibly skeletal
-actin gene regulation is not responsive to the
mechanical component alone in the stretch stimulus but rather is
responsive to growth factor signaling induced during stretch-induced
skeletal muscle hypertrophy. Our current data demonstrate that
pretranslational regulation of skeletal
-actin in skeletal myotubes
is extremely sensitive to the availability of serum and growth factors.
Although serum availability and mechanical stretch interact to
influence skeletal -actin gene regulation, these factors appear to
regulate skeletal
-actin promoter activity by different mechanisms. Increasing the serum content of the culture medium induces skeletal
-actin promoter activity and mRNA concentration in skeletal
myotubes. Increased SRF protein interaction with the SRE1 of the
skeletal
-actin promoter appears to be a mechanism by which skeletal
-actin promoter activity is induced by serum and growth factors. SRF is a member of the MADS
(MCM1-agamous-Arg-80-deficiens-SRF) box family of transcription factors
and binds to the SRE CC(A/T)6GG (CArG box) as a homodimer. SRF has been shown to be important for
myoblast proliferation and differentiation (18, 32). Stretch responsiveness to the c-fos promoter
in cultured cardiac myocytes involves SRF interaction with the
c-fos SRE (25). Plasmid DNA injections
into the chicken ALD muscle have demonstrated that SRE1 is important
for induction of skeletal
-actin promoter activity during skeletal
muscle hypertrophy in the animal (2, 3). Additionally, the SRF-SRE1
binding complex demonstrates an altered mobility in nuclear extracts
from the stretched ALD muscle of wing-weighed birds, compared with
control ALD muscle nuclear extracts (2). Phosphorylation and protein
dimerization with other nuclear regulatory proteins are two mechanisms
providing the specificity of action of SRF (4). SRF contains multiple
serine/threonine residues in its dimerization, DNA binding, and
catalytic domains (14), and the binding affinity of SRF for DNA in
vitro can be altered by its phosphorylation state (20). However, our
data suggest that serum availability increases SRF-SRE1 interactions in
skeletal myotube cultures by increasing the concentration of SRF protein.
Differentiated skeletal myotubes maintained under growth-limiting
conditions had a decreased concentration of skeletal -actin mRNA,
and the addition of static stretch under these conditions further
reduced the concentration of skeletal
-actin mRNA. The activity of
the skeletal
-actin promoter was also repressed under these
conditions, and the first 100 bp of the promoter were sufficient for
stretch-induced repression. Although we have demonstrated that serum
status alters SRF-SRE1 binding interactions, mechanical stretch did not
affect SRF-SRE1 binding interactions or the concentration of SRF
protein. Deletions and mutations of the
99-bp skeletal
-actin
promoter revealed that the TEF-1 element (
66) was necessary for
stretch-induced repression of skeletal actin promoter activity. However, the TEF-1 repression mechanism appears to have serum dependence, since no stretch effect was seen in cells maintained in G/D
medium. The mechanism of TEF-1 involvement in the stretch-induced repression of promoter activity did not appear to be due to an altered
quantity of binding interactions with the skeletal
-actin promoter.
Therefore stretch-induced repression of skeletal
-actin promoter activity occurs through a different mechanism from serum dependence.
Our data imply that the stretch-induced repression of skeletal
-actin promoter activity in skeletal myotubes was mediated through a
posttranslational modification and/or altered protein-protein interactions of the TEF-1 protein. Others have noted that the TEF-1
binding site (5'-CATTCCT-3') is not necessary for basal or
tissue-specific activity of the skeletal
-actin promoter in skeletal
myotubes (5). However, the TEF-1 element is important for the
regulation of other muscle-specific genes, including
-MHC (29),
cardiac troponin T (22), and
-cardiac MHC (23) in cardiac myocytes.
TEF-1 has been implicated in the transmission of
1-adrenergic signaling in
cultured cardiac myocytes. Protein kinase C (PKC)-
is upregulated in
cardiac myocytes induced to hypertrophy by
1-adrenergic stimulation and
mechanical stretch (25). The
-MHC promoter requires a TEF-1 site for
1-adrenergic induction in
cardiac myocytes (15).
1-Adrenergic stimulation of the
skeletal
-actin promoter in cardiac myocytes works through the SRE1
and TEF-1 elements (16). The PKC-
pathway appears to be an example
of a signaling cascade that can alter promoter activity through the
TEF-1 element. The signaling cascade that could posttranslationally
modify the TEF-1 protein and repress skeletal
-actin promoter
activity in stretched skeletal myotubes remains to be determined.
Differences in skeletal -actin regulation due to alterations in
serum availability and mechanical stretch were demonstrated between
cultured cardiac myocytes and skeletal myotubes. Stretched cardiac
myocytes have been shown to increase skeletal
-actin mRNA
concentration (27). Our current data extend this finding by
demonstrating that the stretch induction of skeletal
-actin mRNA is
dependent on serum availability. Cardiac cultures maintained in G-L
medium had an increased skeletal
-actin mRNA concentration compared
with cells maintained in G/D medium, and mechanical stretch in G-L
medium further induced skeletal
-actin mRNA levels. The effect of
culture medium serum content on the stretch induction of skeletal
-actin mRNA may also be affected by cardiocyte contractility. Serum-free culture conditions can inhibit cardiomyocte contractility, and our current results cannot rule out interaction between decreased contractile activity and mechanical stretch as a mechanism for altering
skeletal
-actin mRNA. However, Simpson et al. (30) demonstrated that
static stretch of cardiac myocytes suppressed the loss of total
myofibrillar protein, MHC, actin, and desmin in both beating and
nonbeating cardiac myocytes. In differentiated skeletal myoblasts,
concentration of skeletal
-actin mRNA was also altered by G-L medium
and further altered by static-stretch in this condition; however, the
effect was a repression rather than the induction seen in
cardiomyocytes. These results demonstrate that the interaction of
stretch and serum does not elicit similar effects on skeletal
-actin
pretranslational regulation in skeletal and cardiac cultures. These
data suggest that the signaling cascades related to the induction of
genes during cardiac myocyte hypertrophy may not be physiologically
relevant to stretch-induced hypertrophy of skeletal muscle.
The 424-bp skeletal
-actin promoter has been shown to be
sufficient for developmental and cell type regulation in skeletal myoblast culture (1). The
424-bp skeletal
-actin promoter is
also sufficient to confer stretch induction during in vivo hypertrophy
of skeletal muscle (3). Additionally, the first 100 bp of the skeletal
-actin promoter are sufficient to confer TGF-
and
-adrenergic
induction in cardiac myocytes (16, 19). In the current study, induction
of the
424-bp skeletal
-actin promoter in stretched cardiac
myocytes is not correlated with stretch-induced increase in skeletal
-actin mRNA concentration. Promoter activity was elevated during
conditions in which skeletal
-actin mRNA was not induced, and
promoter activity was not induced during conditions in which skeletal
-actin mRNA was increased. The lack of change in promoter activity
when the corresponding mRNA is induced by stretch in cultured cardiac
myocytes was previously reported for other cardiac "developmental
reexpression" genes (27). Stretched cardiac myocytes increase the
mRNA concentration for atrial natriuretic factor (ANF) and
-MHC;
however, activity from the ANF or
-MHC promoters has been reported
to not increase during stretch (27). The mechanisms of gene regulation
critical for the reexpression of developmental genes, such as skeletal
-actin, during stretch-induced hypertrophy of cardiac myocytes remain to be determined. The gene regulation involved in cardiomyocyte stretch induction of developmental genes appears to be a complex phenomenon.
Very possibly, in vivo gene regulation in stretched skeletal muscle may
be different from that described for in vitro stretch of skeletal
myotube and cardiac myocyte cultures. However, these current results
demonstrate that skeletal -actin gene expression is responsive to
stretch in cultured striated muscle cells and that this response is
modulated by the availability of serum and growth factors. Mechanical
stretch in the absence of serum decreases skeletal
-actin mRNA
concentration in skeletal myotubes and increases skeletal
-actin
mRNA concentration in cardiac myocytes. Serum availability and stretch
both alter skeletal
-actin mRNA concentration and promoter activity
in skeletal myotubes, but, at the level of the skeletal
-actin
promoter, these stimuli invoke regulation through different mechanisms.
SRF-SRE1 interactions in skeletal myotubes appear to be influenced by
the serum concentration, whereas stretch-induced repression needs an
intact TEF-1 binding site. These results support the conclusion that
stretch and serum interact to alter skeletal
-actin regulation
pretranslationally in cultured cardiac and skeletal muscle cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Robert J. Schwartz for generosity in supplying probes.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants RO1-AR-19393 and F32-AR-8328.
Present address of James A. Carson: Dept. of Cell Biology, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: F. W. Booth, Dept. of Integrative Biology, Pharmacology and Physiology, University of Texas Health Science Center, 6431 Fannin St., Houston, TX 77030.
Received 2 March 1998; accepted in final form 19 August 1998.
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