(Received for publication, November 7, 1994)
From the
Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/µg of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.
Insulin-like growth factors (IGFs) ()are potent
mitogens involved in stimulating skeletal muscle
growth(1, 2, 3, 4) . They increase
amino acid uptake and protein synthesis, decrease protein degradation,
and stimulate the proliferation and differentiation of skeletal muscle
cells(2, 5, 6, 7, 8, 9, 10) .
IGF's have been shown to be secreted from several mammalian
skeletal muscle cell lines(8, 11, 12) . A
number of studies have revealed that IGF-2 is released during myoblast
proliferation while IGF-1 efflux is observed during skeletal muscle
differentiation (1, 8) . Increases in IGF-1 mRNA have
been observed during muscle regeneration after
injury(13, 14, 15) , and during work-induced
compensatory hypertrophy(16) . It has been suggested that the
increased secretion of IGF-1 during work-induced hypertrophy (16) may promote the accumulation of proteins in skeletal
muscle cells by an autocrine mechanism but the level of IGF-1 release
from skeletal muscle cells undergoing hypertrophy is not known.
The mitogenic effects of insulin-like growth factors are regulated by their binding proteins (reviewed in (17, 18, 19, 20) ). IGF binding proteins are released from cells which also secrete insulin-like growth factors (1, 18, 21, 22) . They have been well characterized in serum in vivo(23) and in conditioned medium from tissue-cultured fibroblasts, liver cells, smooth muscle, decidual cells, and mammalian skeletal myoblasts (reviewed in Refs. 1, 18, 21, and 22). The efflux of IGF binding proteins from these cultured cells correlates with changes in the secretion of IGF-1. Thus, during C2 skeletal muscle cell line differentiation, increased secretion of IGF-1 is accompanied by increased release of IGF binding proteins(1) . There are no reports on IGF binding protein efflux during either skeletal muscle repair or skeletal muscle hypertrophy.
This study was conducted to first establish whether primary cultures of differentiated avian skeletal muscle cells secrete IGF-1 and IGF binding proteins in a manner similar to tissue-cultured mammalian skeletal muscle cell lines. Second, using blocking antibodies, we determined whether IGF-1 secreted by the muscle cells could act as a autocrine/paracrine growth stimulator. Third, we determined the effect of repetitive mechanical stimulation on the sensitivity of the cells to exogenous IGF-1. Finally, the effect of mechanical stimulation on the autocrine secretion of IGF-1 and IGF binding proteins from the cultured avian pectoralis muscle cells was examined. The results indicate that IGF-1 is an autocrine/paracrine growth factor in differentiated avian pectoralis skeletal muscle cultures. Repetitive mechanical stimulation of the muscle cells increased the sensitivity of the cells to exogenous IGF-1, and acutely stimulated IGF-1 release; but it had no long-term effect on either IGF-1 or IGF binding protein release. In contrast, the release of IGF-1 and IGF binding proteins from the muscle cells was dramatically stimulated by embedding the cells in a three-dimensional collagen type I matrix after myofiber formation. This stimulated release of IGF-1 by type I collagen may be responsible for its ability to stimulate skeletal myofiber growth in vitro(24) .
Figure 1: Cell stretch/relaxation activity (TRIAL39.PGM). Differentiated skeletal muscle cells were mechanically stimulated with five 12% stretches and relaxations of the substratum over a 20-s period followed by a 10-s rest period. The pattern was repeated twice more, followed by a 30-min rest period.
Statistical analyses of the data were performed by t tests for unpaired values using a statistical software program (SIGMASTAT, Jandel Scientific).
Figure 2: Effect of IGF-1 and insulin on protein/DNA ratio (A), protein synthesis (B and C), and myosin content (D) in control and mechanically-stimulated skeletal muscle cells. Cultures in A and D were mechanically stimulated for 48 h, while those in B and C were for 2 h, in serum-free MM medium containing the level of exogenous IGF-1 or insulin indicated on the x axis. Values are expressed as the mean ± S.E. of 4-6 samples and compared by unpaired t test.
Figure 3:
Effect of collagen on IGF-1 release from
skeletal muscle cells grown on plastic tissue culture dishes, or on
silicone rubber membranes. On day 3 postplating, cultured muscle cells
were fed either fresh 85/10/5 medium or embedded in a collagen gel
matrix. On day 6 the cells were rinsed and incubated in defined-serum
free medium. Conditioned media were collected for the 0-24 and
24-48 h time periods, and analyzed for IGF-1. The values
represent the mean ± S.E. of five to eight samples, and are
compared using the unpaired t test. NCE, noncollagen
embedded; CE, collagen embedded. , NCE, plastic;
, NCE, silicone; &cjs2112;, CE, plastic; &cjs2110;, CE,
silicone.
Within the same cell preparation, IGF-1 release was always greater when the muscle cells were grown on the elastic membranes of the mechanical cell stimulator compared to plastic culture dishes. Thus, noncollagen-embedded skeletal muscle cells grown on elastic membranes released 2.6-fold more IGF-1 after 24 h, and 7.8-fold more after 48 h, compared to cells on rigid plastic dishes (Fig. 3). When embedded in a collagen matrix, the muscle cells growing on the elastic membranes released 1.3- and 1.8-fold more IGF-1/µg of protein after 24 and 48 h of incubation in defined medium, respectively, compared to those grown on plastic culture dishes (Fig. 3). In subsequent experiments, controls were therefore always run with the same cell preparation growing on identical substrata.
To ascertain whether the increased IGF-1 found in
conditioned medium from collagen-embedded cells was trapped within the
collagen gels from prior incubation with serum and chicken embryo
extract containing medium, collagen gels were prepared in 4-well plates
with 85/10/5 medium, but without cells, and treated the same way as the
muscle cell cultures. After rinsing the gels by the normal protocol,
they were incubated in serum-free medium for a 24-h period, and
conditioned medium collected for IGF-1 analysis. The collagen gels
without cells released an average of 381 ± 42 pg of
IGF-1/well/24 h, compared to 1,790 ± 270 pg of IGF-1/well/24 h
observed in conditioned medium from collagen-embedded cells grown in
plastic culture plates. To further examine this question, the amount of
IGF-1 trapped from 85/10/5 medium in collagen gels in the presence of
skeletal muscle cells was determined by preparing the collagen gels
with medium containing tracer levels of I-IGF-1. Fresh
medium containing tracer levels of
I-IGF-1 was added to
the cultures every 24 h. The 6-7-day-old cultures were then
rinsed by the normal protocol, and the release of radioactivity
measured over a 24-h period. The rinsed muscle cells embedded in the
collagen matrix released 6.88% of the total initial medium
radioactivity over a 24-h period. This equaled 42 pg of IGF-1/well
trapped by the collagen gels, 10-15-fold less than the IGF-1
released from collagen-embedded cells into the medium during this time
period. The
I-IGF-1 measured in homogenates of the
collagen-embedded cells from these experiments was 1.9% of the total
radioactivity in the original 85/10/5 medium. These results indicate
that only a small percent of the IGF-1 released into the conditioned
medium resulted from IGF-1 trapped from serum and embryo extract
containing medium.
Figure 4: Effect of stretch on IGF-1 release from skeletal muscle cells. Six-day-old collagen-embedded cells were incubated in defined MM medium, and stimulated mechanically every 30 min as outlined under ``Experimental Procedures.'' Conditioned medium was collected at 0.5, 1, 2, and 3 days after initiating stretch, and IGF-1 released into the medium was measured by radioimmunoassay. Results are expressed as the mean ± S.E. of three to six values per group and compared by t test for unpaired values (p > 0.05 for all control versus stretch groups).
The effect of different patterns of mechanical stimulation on IGF-1 efflux from the collagen-embedded muscle cells was examined next. The cells were mechanically stimulated 6.7-21% every 30 min for 24 h with the same frequency as in TRIAL39.PGM. No significant differences in IGF-1 efflux were observed among the different stretch intensity groups (Fig. 5A). Similarly, a 6-fold increase in the frequency of mechanical stimulation (5-min rest periods, TRIAL52.PGM) showed no effect on the release of IGF-1 from the muscle cells (Fig. 5B).
Figure 5: Effect of stretch intensity and frequency on IGF-1 efflux. Collagen-embedded skeletal muscle cells were switched to defined MM medium from day 6 to day 8 postplating. Cultures in A were mechanically stimulated for 24 h by the same frequency pattern as outlined in Fig. 1, but with varied percent intensities of stretch. This experiment was performed with the same cell preparation by varying prong heights between wells as described under ``Experimental Procedures.'' Cultures in B were mechanically stimulated every 5 min instead of every 30 min by the same pattern of activity as outlined in Fig. 1. Results are expressed as the mean ± S.E. of six values per group and compared by t test for unpaired values.
To examine the time course of IGF-1 efflux with stretch, day 6 noncollagen-embedded cells were mechanically stimulated using the TRIAL39.PGM activity pattern, and conditioned medium was collected at 1, 2, 4, 8, 12, and 24 h of stretch, with fresh medium added to the cultures at each time point. Noncollagen-embedded cultures were used in these kinetic studies to eliminate the collagen as a potential diffusion barrier. While total accumulated release of IGF-1 over the 24-h incubation period (i.e. addition of released IGF-1 at all the time points) was not significantly different in these noncollagen-embedded cultures (control static cultures: 18.7 pg of IGF-1/µg of protein/24 h; stretched cultures: 16.4 pg of IGF-1/µg of protein/24 h), as found for the collagen-embedded culture experiments described above, the kinetics of IGF-1 release was significantly different between control and stretched cells. IGF-1 release from static control cells increased rapidly during the first 4 h and then increased at a slower rate over the remaining 20-h period (Fig. 6). IGF-1 release from stretched cells was significantly increased during the first hour of stretch compared to static controls, reaching a maximum at 4 h of mechanical stimulation (Fig. 6). IGF-1 efflux then declined in these cultures even though mechanical stimulation continued. This pattern of IGF-1 release was observed in three different experiments involving noncollagen-embedded muscle cells, and in four different experiments using collagen-embedded cells (data not shown).
Figure 6: Time course of IGF-1 efflux from noncollagen-embedded skeletal muscle cells. The cells were mechanically stimulated by TRIAL39.PGM. The media was removed at each time point, and fresh defined MM media added. IGF-1 content was assayed in each sample as outlined under ``Experimental Procedures.'' Results are expressed as the mean ± S.E. of 2-3 values and compared by t test for unpaired values.
Figure 7:
Collagen-induced efflux of IGF-1 from
skeletal muscle mixed cultures, skeletal myofiber-enriched, and
fibroblast-enriched cultures. Myofiber-enriched cultures and fibroblast
only cultures were prepared as described under ``Experimental
Procedures.'' Six-day-old cultures were incubated in defined MM
medium for 24-48 h and IGF-1 efflux measured from
noncollagen-embedded (A) and collagen-embedded (B)
cells. Results are expressed as the mean ± S.E. of four values
and compared by unpaired t test. , mixed cultures;
, myofiber-enriched cultures; &cjs2113;, fibroblast-enriched
cultures.
Figure 8: Effect of anti-IGF-1 antibody on protein synthesis in noncollagen-embedded and collagen-embedded skeletal muscle cells. Five-day-old noncollagen-embedded and collagen-embedded skeletal muscle cells were rinsed and preincubated for 48 h in MM medium containing 25 and 250 µg of anti-IGF-1 rabbit antibody, respectively. Control cells were preincubated in MM medium without the antibody for 48 h. Protein synthesis was assayed over a 4-6-h time period, with or without the antibody. Values are expressed as the mean ± S.E. of 8 values and compared by unpaired t test.
Figure 9: Detection of IGF binding proteins released from skeletal muscle cell cultures. Conditioned medium was analyzed for IGF binding proteins using ligand blots as described under ``Experimental Procedures.'' The autoradiography shows three binding proteins of molecular masses 31, 36, and 43 kDa. No significant differences in IGF binding protein levels were detected between control (C) and stretched (S) cultures.
Figure 10: IGF binding proteins released from noncollagen-embedded and collagen-embedded skeletal muscle cells. Differentiated muscle cells were grown either with or without collagen embedding as described under ``Experimental Procedures.'' On day 6 postplating, the cells were rinsed for 2 h and incubated in defined medium for 0-24, and 24-48 h. Conditioned medium was analyzed for binding protein levels by ligand blotting and quantitative densitometric analysis as outlined under ``Experimental Procedures.'' Values (arbitrary density units/µg of cell protein) are expressed as the mean ± S.E. of six samples per group and compared by unpaired t test. NCE, noncollagen embedded; CE, collagen embedded. All statistical analyses were done comparing NCE versus CE in the different groups.
Figure 11: Time course of stretch-induced release of IGF binding proteins. Differentiated skeletal muscle cells were embedded in a collagen gel on day 3 postplating, and mechanically stimulated by TRIAL39.PGM starting on day 6. Conditioned medium was collected at 0.5, 1, 4, 8, and 12 h of stretch, and analyzed for binding proteins by ligand blotting as described under ``Experimental Procedures.'' Values are the mean ± S.E. of 4 samples per group and compared by unpaired t test.
This is the first report assessing the efflux of IGF-1 from differentiated primary avian skeletal muscle cells in tissue culture. This study revealed that primary cultures of well-differentiated skeletal myofibers release IGF-1 in significant amounts. Autocrine secretion of IGF-1 has been hypothesized to be involved in work induced skeletal muscle growth in vivo(16) , and we tested this hypothesis with an in vitro model of stretch-induced skeletal muscle growth. Mechanical stretch influenced the sensitivity of skeletal muscle cells to exogenously added IGF-1, and increased the acute but not long-term release of IGF-1 from these cells. On a nanomolar basis, the acute release of IGF-1 with stretch was found to be 20-40-fold less than the amount of recombinant IGF-1 required to stimulate muscle growth in mechanically stimulated cultures in vitro. If the stretch-induced autocrine production of IGF-1 is involved in stretch-induced muscle growth, it must be either more biologically active or more accessible to the IGF-1 receptor than exogenously added recombinant human IGF-1.
The acute secretion of IGF-1 from cultured skeletal muscle cells in response to mechanical stimulation is very similar to the acute, but not long-term, stretch-induced release of atrial natriuretic peptide from cardiac cells(34) . It may result from the release of already synthesized IGF-1, rather than newly synthesized IGF-1. Immunocytochemical studies demonstrate that the cytoplasm of myoblasts and newly formed myotubes contains increased IGF-1 levels during muscle regeneration in vivo(13, 14, 35) . In the present study, skeletal muscle cells were utilized 3 or 4 days after myofiber formation in vitro, and it is possible that these cells also contain intracellular IGF-1 stores. During the first hour of mechanical stimulation in vitro, differentiated skeletal muscle cells appear to be partially damaged, based on temporary creatine kinase release and protease activation in the stretched skeletal muscle cells (25) . The partial damage to the muscle cells by stretch could result in the release of the intracellular IGF-1, as part of a repair process.
Differentiated
avian pectoralis muscle cells were found to secrete not only IGF-1 but
also IGF binding proteins of molecular masses 31, 36, and 43 kDa. This
is the first report on the secretion of IGF binding proteins from
differentiated avian skeletal muscle cells. A number of studies have
shown the presence of IGF binding proteins in human and chicken
serum(18, 33) , and human amniotic fluid(21) ,
as well as in conditioned medium of tissue cultured liver
cells(18) , and mammalian muscle cell
lines(1, 22) . The C muscle cell line
secretes a single IGF binding protein of 29 kDa(22) , while the
C
C
cell line releases three binding proteins
of molecular masses 24, 30, and 32 kDa(1) . The three IGF
binding proteins released from the primary avian skeletal muscle cells
are similar in molecular mass to the binding proteins found in avian
serum in vivo (28, 33, and 41 kDa)(33) . Mechanical
stimulation of the skeletal muscle cells had no significant effect on
the efflux rate of IGF binding proteins at any of the time periods
studied.
A second significant finding in this study was the increased release of IGF-1 and IGF binding proteins from skeletal muscle cells after embedding them in a three-dimensional type I collagen matrix. Collagen-embedded cells released 3-11 times more IGF-1 than noncollagen-embedded cells. There is evidence that IGF-1 stimulates collagen synthesis (36) but there appear to be no studies on the effect of collagen on IGF-1 release. Embedding the myofibers in a collagen gel matrix stimulates their hypertrophy(24, 37) , possibly by activating IGF-1 synthesis and secretion as a paracrine/autocrine growth factor. The mechanism by which collagen enhances IGF-1 release from avian pectoralis muscle cells is not known. In differentiating hepatocytes, collagen promotes the activity of transcription factors resulting in the increased transcription of serum protein genes, such as albumin (38, 39) . Collagen may interact with cell surface receptors resulting in increased transcription of the IGF-1 gene. Because collagen type I recognizes and binds to integrins(40, 41, 42) , the effects of collagen on IGF-1 expression may be modulated via these receptors.
In addition to the differences in IGF-1 efflux from noncollagen-embedded and collagen-embedded cells, skeletal muscle cells grown on a silicone rubber substratum consistently released greater amounts of IGF-1 into the conditioned medium than when grown on plastic culture plates. These results indicate the importance of running proper controls of cells growing on identical substratum. The elastic substratum may have greater permeability than polystyrene plastic to gases such as oxygen and carbon dioxide, resulting in increased cellular activities and leading to elevated levels of IGF-1 production. Skeletal muscle hypoxia not only reduces muscle mass but also reduces oxidative metabolism in the muscle tissue(43) .
The tissue cultures utilized in these experiments consisted of two main cell types, myofibers and fibroblasts. Lowe et al.(44) reported that fibroblasts are capable of synthesizing IGF-1 in vivo. Our experiments using enriched myofiber or confluent fibroblast cultures showed that both cell types are capable of releasing IGF-1. Whereas in mouse primary skeletal muscle cultures the muscle cells produce greater amounts of IGF-1 than fibroblasts(11) , the avian fibroblasts released greater amounts of IGF-1 than the enriched myofiber cultures on a microgram cellular protein basis. But, since 80-90% of the cellular protein in the mixed avian muscle cultures utilized in this study arises from skeletal myofibers(30) , the production of IGF-1 by the myofibers in these cultures on a microgram cell protein basis constitutes the major part of total IGF-1 release. It is difficult, however, to determine the exact contribution of each cell type in the mixed cultures since the two cell types appear to interact in regulating total IGF-1 efflux in a complex manner when co-cultured (Fig. 7), as found previously for the regulation of total protein degradation in the two cell types(45) . IGF-1 secretion in mixed cultures was less than in either cell type alone, indicating some form of feedback inhibition when the two cell types are cultured together.
IGF-1 secreted from cultured skeletal muscle cells can be
considered an important autocrine factor. Our experiments showed that
protein synthesis rates are significantly reduced in the muscle cells
when incubated in the presence of anti-IGF-1 antibody. Similarly,
[H]thymidine uptake in fetal rat myoblasts was
blocked when these cells were incubated with a monoclonal antibody
against human somatomedin(12) . Locally produced IGF-1
therefore plays an important role in the maintenance of tissue-cultured
skeletal muscle cells due to its effects on anabolic processes.
In summary, this paper shows that IGF-1 and IGF binding proteins are released from differentiated avian pectoralis muscle cell cultures, and that the long-term in vitro release of these proteins from the muscle cells is not significantly stimulated by stretch. Stretch-induced myofiber hypertrophy in cultured skeletal muscle cells may involve the short-term increase in IGF-1 secretion, changes in IGF-1 receptors, or a non-IGF-1-related mechanism. In addition, significant collagen-induced IGF-1 and IGF binding protein release from the differentiated muscle cells occurs in vitro. Further studies are needed to examine the mechanisms leading to collagen-induced IGF-1 and IGF binding protein synthesis and/or release from skeletal muscle cells.