Effect of serum and mechanical stretch on skeletal alpha -actin gene regulation in cultured primary muscle cells

James A. Carson and Frank W. Booth

Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Health Science Center, Houston, Texas 77030

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of this study was to determine whether mechanical stretch or serum availability alters pretranslational regulation of skeletal alpha -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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -actin is a primary component of skeletal muscle thin filament and is expressed after myoblast differentiation. Skeletal alpha -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 alpha -actin mRNA between the third and sixth day of stretch (3), and skeletal alpha -actin promoter activity is induced at 6 days of stretch (2). In cardiac muscle, skeletal alpha -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 alpha -actin gene expression is induced both in the animal (28) and in culture (27). Skeletal alpha -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 alpha -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 alpha -actin gene regulation in differentiated skeletal myotubes has not been investigated. Furthermore, the interaction of serum and passive stretch on skeletal alpha -actin gene regulation in skeletal myotube and cardiac myocyte cultures has not been determined.

It is not known whether stretch induces skeletal alpha -actin mRNA expression in skeletal myotubes or whether there is any stretch-induced regulation at the level of the skeletal alpha -actin promoter. Stretching cardiac myocytes cultured in serum-free medium induces c-fos and skeletal alpha -actin mRNA concentrations, and the stretch responsiveness of c-fos is regulated at the level of the promoter (25, 27). Skeletal alpha -actin promoter regulation has not been investigated in stretched cardiac myocytes. Skeletal alpha -actin promoter regulation has been examined in cardiac myocytes induced to hypertrophy by transforming growth factor-beta (TGF-beta ) and alpha -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 alpha -actin in cultured striated muscle cells. A second purpose of the study was to compare the stretch responsiveness of skeletal alpha -actin expression in two types of striated muscle lineages, skeletal and cardiac myocytes.

We demonstrate that serum availability and stretch regulate skeletal alpha -actin pretranslationally in skeletal myotubes and cardiac myocytes. In skeletal myotubes, serum availability and stretch use different regulatory mechanisms to decrease skeletal alpha -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 alpha -actin mRNA concentration of cardiac myocytes is altered by the serum content of the culture medium; skeletal alpha -actin mRNA is increased during stretch in low-serum conditions. These results demonstrate that skeletal alpha -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 alpha -actin differently in these two types of striated muscle.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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 alpha -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 alpha -actin SRE1 double-stranded oligonucleotide for mobility shift experiments comprised nucleotides -100 to -73 (5'-GCCCGACACCCAAATATGGCGACGGCCG-3') of the skeletal alpha -actin promoter. The skeletal alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Skeletal alpha -actin mRNA concentration in skeletal myotubes. The concentration of skeletal alpha -actin mRNA in skeletal myotubes was altered by the concentration of serum in the culture medium. Skeletal alpha -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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Fig. 1.   Serum availability and mechanical stretch alter skeletal alpha -actin (SkA) mRNA concentration in skeletal myotubes. Northern analysis of 10 µg of total RNA. A: SkA mRNA concentration in control skeletal myotube cultures in growth-limiting (G-L; n = 8) medium or in growth/differentiation medium (G/D; n = 9). B: SkA mRNA concentration in control (n = 8) and stretched (16%, 16 h; n = 8) skeletal myotubes switched to G-L medium. SkA values were corrected for 18S rRNA integrated optical density (IOD; see METHODS) on same membrane. Values are means ± SE. * Significantly different from control of same medium treatment group (P <=  0.05); + control G-L skeletal myotubes significantly different from control G/D skeletal myotubes (P <=  0.05).

The stretch responsiveness of skeletal alpha -actin mRNA in skeletal myotubes was examined after several stretch paradigms. Skeletal myotubes were stretched for 8, 16, and 24% of basal membrane support length. Stretch did not induce any change in skeletal alpha -actin mRNA concentration for skeletal myotubes maintained in G/D medium (Fig. 2A). Skeletal myotubes maintained in G-L medium decreased skeletal alpha -actin mRNA levels 26% (P = 0.032; Figs. 1B and 2B) in response to 16 h of 16% static stretch. This decrease in skeletal alpha -actin mRNA was transient returning to control levels at 42 h poststretch (data not shown).


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Fig. 2.   SkA mRNA levels in skeletal myotubes and SkA and serum response factor (SRF) mRNA levels in cardiac myocytes. A: Northern analysis of 10 µg of total RNA from skeletal myotube cultures maintained in G/D medium: control (C) and 8, 16, and 24% stretch of initial membrane length for 16 h (n = 5 for each group). B: Northern analysis of 10 µg of total RNA from skeletal myotube cultures maintained in G-L medium: control and 16% stretch (S) of initial membrane length for 16 h (n = 8 for each). See Fig. 1 for tabulated results. C: Northern analysis of 10 µg of total RNA from control and stretched cardiac myocyte cultures maintained in G-L medium (n = 4 for each). See Fig. 3 for tabulated results.

The primary skeletal myotube cultures in the current study contained a mixture of primary skeletal myoblasts and fibroblasts (1, 5, 7). Primary fibroblast cultures have been shown to be sensitive to serum, the number of fibroblasts being depleted after exposure to low serum conditions (27). To demonstrate that the different stretch responses of skeletal alpha -actin mRNA concentration in skeletal myotubes maintained in either G/D or G-L medium were not due to fibroblast depletion, skeletal myotube cultures maintained in G/D medium were depleted of fibroblasts with cytosine arabinoside. There was no effect of stretch on the skeletal alpha -actin mRNA concentration in skeletal myotube cultures maintained in G/D medium and depleted of fibroblasts. Under these conditions, skeletal alpha -actin mRNA expression was not different between control (0.483 ± 0.091 IOD) and 8% (0.516 ± 0.077 IOD), 16% (0.543 ± 0.013 IOD), or 24% (0.463 ± 0.091 IOD) static stretch. Thus the mRNA level of skeletal alpha -actin in stretched skeletal myotubes maintained in G/D medium was independent of the presence of fibroblasts, and conversely these data demonstrate that fibroblast depletion alone was not sufficient to cause the stretch-induced reduction of skeletal alpha -actin mRNA in skeletal myotubes maintained in G-L medium.

The decreased skeletal alpha -actin mRNA concentration was not due to changes in the total RNA pool. Total RNA was not different between control and stretched skeletal myotubes maintained in G-L medium (10.3 ± 1.4 vs. 11.8 ± 1.3 µg RNA/dish, respectively; n = 8) or G/D medium (37.3 ± 2.7 vs. 41.4 ± 3.0 µg RNA/dish, respectively; n = 8 and 10, respectively). Total RNA content increased 262% (P = 0.00001) in control skeletal myotubes maintained in G/D medium (37.3 ± 2.7 µg/dish; n = 8) compared with control skeletal myotubes switched to G-L medium (10.3 ± 1.4 µg/dish; n = 8).

Skeletal alpha -actin mRNA concentration in cardiac myocytes. Skeletal alpha -actin mRNA levels were also examined in cardiac myocytes. Unlike the effect of serum increasing skeletal alpha -actin mRNA concentration in skeletal myotubes, skeletal alpha -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 alpha -actin mRNA was previously shown to increase during stretch of cultured myocytes (27). Our current study extends these findings by reporting that skeletal alpha -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 alpha -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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Fig. 3.   Serum availability and mechanical stretch alter SkA and SRF mRNA concentration in cardiac myocytes. Northern analysis of 10 µg of total RNA. A: cardiac myocyte SkA mRNA concentration in control and stretched (20%, 7 h) cells maintained in either G-L medium (n = 4 control, 4 stretched) or G/D medium (n = 4 control, 4 stretched). B: SRF mRNA concentration in control and stretched (20%, 7 h) cardiac myocytes maintained in either G-L medium (n = 4 control, 4 stretched) or G/D medium (n = 4 control, 4 stretched). SkA and SRF values were corrected for 18S rRNA IOD on same membrane. Values are means ± SE. * Significantly different from control of same medium treatment group (P <=  0.05); + control G-L skeletal myotubes significantly different from control G/D skeletal myotubes (P <=  0.05).

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 alpha -actin mRNA was not induced, and SRF mRNA was not significantly elevated by stretch under conditions in which skeletal alpha -actin mRNA was increased. These data suggest that induction of SRF and skeletal alpha -actin mRNAs are not correlated during static stretch of cardiac myocytes.

Skeletal alpha -actin promoter analysis. Transient transfection assays were used to examine skeletal alpha -actin promoter regulation during the stretch of striated muscle. The -424-bp skeletal alpha -actin promoter is sufficient for tissue-specific and developmental regulation in cultured myocytes (1). The activity of the -424 skeletal alpha -actin promoter generally followed the mRNA expression pattern of skeletal alpha -actin. Serum availability altered skeletal alpha -actin promoter activity in differentiated skeletal myotubes. The -424 skeletal alpha -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 alpha -actin promoter between control and stretch cultures in G/D medium for any stretch paradigm employed (data not shown). Stretch decreased skeletal alpha -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 alpha -actin promoter activity 34% (P = 0.006) in stretched cultures relative to control cultures (Fig. 4B). Deletions of the skeletal alpha -actin promoter revealed that the -99-bp and -77-bp promoters were sufficient for this stretch-induced repression of skeletal alpha -actin promoter activity (Fig. 4C). A skeletal alpha -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 alpha -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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Fig. 4.   Serum availability and mechanical stretch alter SkA promoter activity in skeletal myotubes. A: -424-bp SkA promoter activity in control skeletal myotube cultures switched to G-L medium (n = 9) or maintained in G/D medium (n = 8). B: -424-bp SkA promoter activity in control (n = 15) and stretched (n = 12; 16%, 16 h) skeletal myotubes switched to G-L medium. C: -99-bp wild-type (WT) SkA (-99 SkA WT; n = 12 Control, 14 Stretch) and -77-bp wild-type (-77 SkA WT; n = 8 Control, 10 Stretch) deletions of -424-bp SkA promoters transfected in skeletal myoblast cultures switched to G-L medium and subjected to stretch (16%, 16 h); -99-bp deletion contained serum response element 1 (SRE1) and transcriptional enhancer factor 1 (TEF-1) elements, whereas -77-bp deletion removed SRE1. Activity of -99-bp SkA promoters containing mutations to either SRE1 (-99 SkA M15; n = 6 Control, 6 Stretch) or TEF-1 (-99 SkA M11; n = 6 Control, 6 Stretch) were also measured. Values are expressed relative to -99 SkA WT SkA expression in control skeletal myotube culture. Values are means ± SE. RLUs, relative light units (see METHODS). * Significantly different from control of same medium treatment group (P <=  0.05); + control G-L skeletal myotubes significantly different from control G/D skeletal myotubes (P <=  0.05).

Skeletal alpha -actin promoter activity was also examined in transiently transfected cardiac myocytes. Although serum availability altered skeletal alpha -actin mRNA concentration in control cardiac myocytes, the -424-bp skeletal alpha -actin promoter transfected into cardiac myocytes was not sensitive to the serum availability (Fig. 5). This is unlike skeletal myotube cultures, in which skeletal alpha -actin mRNA and -424-bp promoter activity paralleled each other. There was not a significant difference between control cultures maintained in G/D medium and G-L medium. The stretch-induced activity of the -424 skeletal alpha -actin promoter in cardiac myocytes did not follow the stretch-induced changes seen in skeletal mRNA levels. Although cardiac myocytes maintained in G-L medium had a stretch-induced increase in skeletal alpha -actin mRNA concentration (Fig. 3A), -424 promoter activity at 6-8 h poststretching was not different from controls (Fig. 5). Promoter activity was also measured 14 h poststretching, and there was also no difference between control and stretched cultures maintained in G-L medium then (data not shown). Cardiac myocytes maintained in G/D medium did not demonstrate a stretch-induced increase in skeletal alpha -actin mRNA levels (Fig. 3A); however, -424 skeletal alpha -actin promoter activity was significantly induced at 6-8 h after static stretch in G/D medium (Fig. 5). By 21 h poststretch, -424 skeletal alpha -actin promoter activity was not different between control (244,754 ± 36,082 RLU; n = 5) and stretched (211,843 ± 33,729 RLU; n = 4) cardiac myocytes maintained in G/D medium.


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Fig. 5.   In stretched cardiac myocytes, induction of SkA promoter activity is dependent on serum availability: -424 SkA promoter activity in control and stretched (20%, 6-8 h) cardiac myocyte cells maintained in either G/D medium (n = 4 Control, 4 Stretch) or G-L medium (n = 4 Control, 4 Stretch). Plasmid containing the cytomegalovirus promoter directing the expression of galactosidase was cotransfected along with actin plasmid DNA to correct for transfection efficiency. Values are means ± SE. * Significantly different between control and stretch of same culture medium treatment group (P <=  0.05).

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 alpha -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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Fig. 6.   Gel mobility shift assays using skeletal myotube cell extracts demonstrate a serum dependence of SRF binding to SRE1 from SkA promoter. Gel mobility shift assays were performed with 5 µg of skeletal myotube whole cell protein extract and end-labeled SRE1 oligonucleotide from SkA promoter. Left: SRF and yin yang 1 (YY1) binding to SRE1 in cells maintained in G-L and G/D medium. Representative of 4 G-L and 4 G/D observations. Right: specificity of binding complexes was demonstrated by adding competitor SRE1, a 100-fold molar excess of unlabeled SRE1 added to binding reaction. Supershifted SRF binding complexes were demonstrated by addition of anti-SRF serum to binding reaction. Supershifted complexes were also formed against YY1 binding complexes using anti-YY1 serum (data not shown).

TEF-1 protein binding complex with the actin TEF-1 binding site did not differ between the control cultures maintained in G/D and G-L medium (Fig. 7). Additionally, there was no alteration in binding at TEF-1 with static stretch, regardless of culture conditions. Although the stretch-induced repression of the skeletal alpha -actin promoter in skeletal myotubes maintained in G-L medium appears to be mediated through the TEF-1 binding site (Fig. 4C), there appears to be no altered binding with stretch. These data suggest that posttranslational modifications of TEF-1 and/or altered protein-protein interactions, rather than altered binding affinity, may be responsible for the stretch-induced repression of the skeletal alpha -actin promoter in skeletal myotubes.


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Fig. 7.   TEF-1 binding is not altered by serum availability and/or mechanical stretch in differentiated skeletal myotube cell extracts. Gel mobility shift assays were performed with 5 µg of skeletal myotube whole cell protein extract and end-labeled TEF-1 oligonucleotide from SkA promoter. Control and stretched (16%, 16 h) whole cell extracts from skeletal myotubes were maintained in either G/D or G-L medium. Data are representative of 4 control and 4 stretched skeletal myotube cultures maintained in G/D medium and 4 control and 4 stretched skeletal myotube cultures maintained in G-L medium. Specificity of binding complexes was demonstrated by adding competitor TEF-1, a 100-fold molar excess of unlabeled TEF-1 added to binding reaction. P, probe.

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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Fig. 8.   SRF protein concentration is altered by serum availability in whole cell extracts from differentiated skeletal myotubes. Western analysis of SRF protein from control and stretched skeletal myotubes maintained in either G/D or G-L medium. See RESULTS for tabulated values. Total protein (50 µg) was added to each lane.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study demonstrates that the pretranslational regulation of skeletal alpha -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 alpha -actin regulation pretranslationally, and this pretranslational regulation was demonstrated at the levels of mRNA concentration and promoter activity. Skeletal alpha -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 alpha -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 alpha -actin mRNA in stretched cardiac myocytes under the same conditions. There was no stretch effect on skeletal alpha -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 alpha -actin mRNA concentration when serum and growth factors are present in abundance.

The lack of skeletal alpha -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 alpha -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 alpha -actin mRNA concentration increases to control levels (3) and skeletal alpha -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 alpha -actin in skeletal myotubes. These facts, taken together with animal data, bring forward the question of whether stretch is sufficient to induce skeletal alpha -actin mRNA expression in skeletal muscle.

A mechanical stretch stimulus may require additional cofactors and/or growth factor signaling to induce skeletal alpha -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 alpha -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 alpha -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 alpha -actin gene regulation, these factors appear to regulate skeletal alpha -actin promoter activity by different mechanisms. Increasing the serum content of the culture medium induces skeletal alpha -actin promoter activity and mRNA concentration in skeletal myotubes. Increased SRF protein interaction with the SRE1 of the skeletal alpha -actin promoter appears to be a mechanism by which skeletal alpha -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 alpha -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 alpha -actin mRNA, and the addition of static stretch under these conditions further reduced the concentration of skeletal alpha -actin mRNA. The activity of the skeletal alpha -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 alpha -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 alpha -actin promoter. Therefore stretch-induced repression of skeletal alpha -actin promoter activity occurs through a different mechanism from serum dependence.

Our data imply that the stretch-induced repression of skeletal alpha -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 alpha -actin promoter in skeletal myotubes (5). However, the TEF-1 element is important for the regulation of other muscle-specific genes, including beta -MHC (29), cardiac troponin T (22), and alpha -cardiac MHC (23) in cardiac myocytes. TEF-1 has been implicated in the transmission of alpha 1-adrenergic signaling in cultured cardiac myocytes. Protein kinase C (PKC)-beta is upregulated in cardiac myocytes induced to hypertrophy by alpha 1-adrenergic stimulation and mechanical stretch (25). The beta -MHC promoter requires a TEF-1 site for alpha 1-adrenergic induction in cardiac myocytes (15). alpha 1-Adrenergic stimulation of the skeletal alpha -actin promoter in cardiac myocytes works through the SRE1 and TEF-1 elements (16). The PKC-beta 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 alpha -actin promoter activity in stretched skeletal myotubes remains to be determined.

Differences in skeletal alpha -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 alpha -actin mRNA concentration (27). Our current data extend this finding by demonstrating that the stretch induction of skeletal alpha -actin mRNA is dependent on serum availability. Cardiac cultures maintained in G-L medium had an increased skeletal alpha -actin mRNA concentration compared with cells maintained in G/D medium, and mechanical stretch in G-L medium further induced skeletal alpha -actin mRNA levels. The effect of culture medium serum content on the stretch induction of skeletal alpha -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 alpha -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 alpha -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 alpha -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 alpha -actin promoter has been shown to be sufficient for developmental and cell type regulation in skeletal myoblast culture (1). The -424-bp skeletal alpha -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 alpha -actin promoter are sufficient to confer TGF-beta and alpha -adrenergic induction in cardiac myocytes (16, 19). In the current study, induction of the -424-bp skeletal alpha -actin promoter in stretched cardiac myocytes is not correlated with stretch-induced increase in skeletal alpha -actin mRNA concentration. Promoter activity was elevated during conditions in which skeletal alpha -actin mRNA was not induced, and promoter activity was not induced during conditions in which skeletal alpha -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 beta -MHC; however, activity from the ANF or beta -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 alpha -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 alpha -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 alpha -actin mRNA concentration in skeletal myotubes and increases skeletal alpha -actin mRNA concentration in cardiac myocytes. Serum availability and stretch both alter skeletal alpha -actin mRNA concentration and promoter activity in skeletal myotubes, but, at the level of the skeletal alpha -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 alpha -actin regulation pretranslationally in cultured cardiac and skeletal muscle cells.

    ACKNOWLEDGEMENTS

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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