From the Department of Biochemistry and Molecular
Biology, Monash University, Clayton, Victoria 3168, Australia and
the ¶ School of Veterinary Science, University of Melbourne,
Parkville, Victoria 3052, Australia
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three members of the family of protease-activated
receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin
receptors. PAR-1 is expressed by primary myoblast cultures, and
expression is repressed once myoblasts fuse to form myotubes. The
current study was undertaken to investigate the hypothesis that
thrombin inhibits myoblast fusion. Primary rodent myoblast cultures
were deprived of serum to promote myoblast fusion and then cultured in
the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells
were apoptotic within 24 h of serum deprivation, whereas less than
15% of thrombin-treated cells showed signs of apoptosis. Proteolysis
was required for the effect of thrombin, but no other serine protease
tested mimicked the action of thrombin. Neither a PAR-1- nor a
PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast
cultures were negative for PAR-3 expression. Myoblasts exposed to
thrombin for 1 h and then changed to medium without thrombin
accumulated apoptosis inhibitory activity in their medium over the
subsequent 20 h. Thus the protective action of thrombin appears to
be effected through cleavage of an unidentified thrombin receptor,
leading to secretion of a downstream apoptosis inhibitory factor. These
results demonstrate that thrombin functions as a survival factor for
myoblasts and is likely to play an important role in muscle development
and repair.
Thrombin is a trypsin-like serine proteinase that has a central
role in hemostasis and thrombosis. The role of thrombin in these
processes is not only dependent on its cleavage of fibrinogen but also
on its ability to regulate the activity of cells such as leukocytes,
platelets, and endothelial cells (1-4). Many of the cellular actions
of thrombin are known to be mediated by a seven-transmembrane domain,
G-protein-coupled receptor
PAR-11 that is activated by a
thrombin cleavage event in its extracellular domain. Proteolysis
generates a new N terminus that acts as a tethered ligand, binding to
another site in the receptor and activating a signal transduction
cascade (5, 6). Results with mice genetically incapable of expressing
PAR-1, however, have shown that not all of the effects of thrombin are
mediated by this receptor, leading to the recent discovery of two
additional thrombin receptors, PAR-3 and PAR-4, which are closely
related to PAR-1 (7-9). Synthetic peptides corresponding to the
tethered ligand sequence of PAR-1 and PAR-4 (but not PAR-3) have been
shown to activate their respective receptor in the absence of
proteolysis (5, 8, 9).
During the process of muscle development, myoblasts proliferate and
then undergo differentiation, fusing to form multinucleated myotubes. A
knowledge of the factors that determine whether myoblasts will undergo
proliferation or differentiation is essential for an understanding of
postnatal growth and repair in skeletal muscle. A number of growth
factors have been shown to influence these processes in cultured
myoblasts. For example, fibroblast growth factor and epidermal growth
factor stimulate myoblast proliferation and inhibit differentiation
(10, 11).
Several lines of evidence have recently indicated that thrombin is an
important regulator of muscle development. Thrombin has been proposed
to play a role in synapse elimination occurring at the neuromuscular
junction during development, because the specific thrombin inhibitor
hirudin blocks synapse elimination both in vitro and
in vivo (12, 13). Prothrombin is expressed in developing
muscle (14), and in muscle cultures transcript levels and thrombin
activity in the medium are increased by cholinergic stimulation (15).
PAR-1 is expressed by cultured myoblasts, but expression is repressed
once myoblasts fuse to form myotubes (16). In addition, recent
immunohistochemical studies in developing muscle indicate that loss of
PAR-1 expression by myoblasts soon after fusion also occurs in
vivo (17). Thrombin was found to cause an increase in the number
of cultured myoblasts, possibly through an increase in proliferation
(16). The aim of the current study was to investigate the ability of
thrombin to influence myoblast differentiation. Expression patterns of
PAR-1 in cultured myoblasts led to the hypothesis that thrombin
inhibits myoblast fusion. Initial experiments were designed to test
this hypothesis, but during the course of these studies it was observed
that thrombin exerted a potent inhibitory effect on myoblast apoptosis.
The results presented here document the effects of thrombin on fusion and apoptosis in primary myoblast cultures.
Materials--
Human Cell Culture--
Primary muscle cell cultures were established
from the hind limb muscles of 2-day-old newborn rats or mice. Cells
were isolated as described, except that the preplating step was carried
out for 1.5 h, and the final Percoll gradient step was omitted
(20). After preplating, nonadherent cells were collected, centrifuged, and resuspended in 10% (v/v) horse serum in Ham's F-10 medium.
For fusion and apoptosis assays, cells were plated at a concentration
of 2 × 105 cells/ml onto Nunc Lab Tek II 8-chamber
glass slides pretreated with poly-L-lysine then coated with
laminin as described (16). Cell suspension (0.5 ml) was added to each
well, and the slides were placed in a humidified atmosphere with 5%
CO2 at 37 °C. Nonadherent cells and other debris were
removed when the medium was changed the following day. Experiments on
myoblasts were performed when cells were approximately 80-90%
confluent, which was usually 2-3 days after plating. At this stage,
fusion to form myotubes was minimal. The medium in the wells was
removed, and the cells were washed three times with Ham's F-10 medium
containing bovine serum albumin (1 mg/ml). The cells were left for 5 min in the last wash before treatment with thrombin or other test
substances in Ham's F-10/bovine serum albumin solution. The cells were
returned to the incubator for various times (24 h unless otherwise
mentioned) before immunocytochemical analysis. In the experiment shown
in Fig. 4B, thrombin-containing medium was removed at
various times, and then the cells were washed and incubated in fresh
serum-free medium for the remainder of the 24 h. The myogenic
nature of the cells was verified by staining with antibodies to myo D1
or desmin (21, 22). Our cultures were shown to be approximately 80% positive for either marker. Rat cells were used for all experiments presented except where particular reagents required the use of mouse
cells, as specified in the text. The same response to thrombin in
apoptosis and fusion assays was observed in mouse cells as in rat cells.
For production of conditioned medium, cells were plated in 6-well
plates and grown until 80-90% confluent before serum deprivation and
treatment with thrombin-containing (100 nM) or control
medium for various times before harvest. Where conditioned medium
contained thrombin, it was inactivated with PPACK until no detectable
thrombin activity remained before being used in apoptosis and fusion
assays. In the experiment shown in Fig. 5, cells were treated for
1 h with thrombin (100 nM) and then washed and exposed
to fresh serum-free medium; conditioned media removed 5, 10, and
20 h after thrombin withdrawal were tested for thrombin activity
and found to contain no detectable activity.
Cell Staining--
Cells were washed gently in Hanks' balanced
salt solution at room temperature, fixed in paraformaldehyde (4% w/v)
in Hanks' balanced salt solution for 10 min, and then washed in PBS
and permeabilized in Triton X-100 (0.5% v/v in PBS). Cultures were blocked for 1 h in PBS containing fetal calf serum (10% v/v) and then incubated in mouse anti-myo D1 (Dako) or anti-desmin (Novocastra) antibodies (both 1:100 in blocking solution) at room temperature for
2 h. The cells were then washed twice in PBS, and primary antibodies were detected with a rhodamine-conjugated rabbit anti-mouse IgG (Dako; 1:400 in blocking solution) for 1 h. Cells were washed twice with 10% fetal calf serum (v/v) in PBS and once in PBS alone and
then stained with the nuclear dye 4',6'-diamidino-2-phenylindole (DAPI)
at 0.1 mg/ml in PBS for 5 min. Slides were mounted in
para-phenylenediamine in glycerol and then examined by
fluorescence microscopy. DAPI was used for the detection of apoptotic
cells on the basis of nuclear morphology; nuclei showing chromatin
condensation and blebbing were classified as apoptotic. Apoptosis was
also detected by an in situ TdT-mediated dUTP nick end
labeling (TUNEL) reaction, performed prior to mounting, using a
commercially available detection kit (Boehringer Mannheim).
Cell Number Analysis--
A Nikon fluorescence microscope linked
to a computer with Microcomputer Imaging Device software (Imaging
Research Inc.) was used for cell counting. Using appropriate
fluorescence filters, separate images of DAPI staining and desmin
staining of a field were captured and superimposed. These dual images
were displayed on the video monitor and used for manual counting of
cells. For apoptosis studies, apoptotic myoblasts were counted and
expressed as a percentage of total myoblasts. For myoblast
differentiation studies, myoblast nuclei in myotubes were counted and
expressed as a percentage of total myoblast nuclei. In each case at
least 100 nuclei/well were counted, and results from triplicate wells were expressed as the means ± standard error. Results were
analyzed using Student's t test.
RNA Preparation and Polymerase Chain Reaction
Analysis--
Total cellular RNA was isolated from rat myoblast
cultures or mouse spleen tissue using (TRI REAGENT, Sigma) according to the manufacturer's instructions. First strand cDNA was synthesized from 7 µg of RNA with Moloney murine leukemia virus reverse
transcriptase using oligo(dT) primer (Ready-To-Go You-Prime
First-Strand Beads, Amersham Pharmacia Biotech). Using the entire
first-strand reaction, polymerase chain reaction amplification was
performed according to the manufacturer's instructions with the
following primer pairs: PAR-1 (intron spanning): sense 5'-ATG GGG CCC
CGG CGC TTG CTG-3', antisense 5'-CCC TAA GCT AGT AGC TTT TTG TAT
ATG-3'; PAR-3: sense 5'-ACA ACA TCC TGT AGC CGG GTC T-3', antisense
5'-TAA CAG AAG ATG ATG ATC ACA-3'; GAPDH: sense 5'-ACC ACC ATG GAG AAG
GCT GG-3', antisense 5'-CTC AGT GTA GCC CAG GAT GC-3'. The samples were
placed in a thermal cycler for 32 cycles of the following profile:
denaturation at 95 °C for 1 min, annealing at 55 °C for 1 min,
and polymerization at 72 °C for 1 min. The polymerase chain reaction
products were electrophoresed in 1.8% (w/v) agarose gels and labeled
with ethidium bromide for photography. Before incubation with mouse
myoblast RNA, PAR-3 primers were validated by the finding that they
produced a band of the appropriate size with mouse spleen RNA, which is known to express PAR-3 (7).
Intracellular Ca2+ Measurement--
For
[Ca2+]i assays, cells were plated in flasks (75 cm2) and grown to confluence then detached with
nonenzymatic dissociation medium (Sigma). [Ca2+]i
was measured at 37 °C using the fluorescent indicator Fura-2
(Molecular Probes) essentially as described by Jenkins et
al. (23) except that the buffer used for loading the cells with
Fura-2 and subsequent assays was 10 mM Hepes buffer, pH
7.4, containing 138 mM NaCl, 5 mM KCl, 1 mM MgCl, 1.5 mM CaCl2, 5 mM D-glucose, and 0.1% bovine serum albumin
(w/v).
Thrombin Inhibits Skeletal Muscle Cell Differentiation--
To
investigate whether thrombin modulates myoblast fusion, rat cells were
grown until almost confluent in serum-containing medium. Cells were
then deprived of serum and cultured in the presence or absence of
thrombin. Cultures were evaluated for myotube formation 12, 24, and
32 h after serum deprivation. In control cultures after 24 h,
35% of myoblast nuclei were present in multinucleated myotubes (Figs.
1, C and D, and
2A). In contrast, fusion was
inhibited in thrombin-treated myoblasts, with less than 10% of
myoblast nuclei present in myotubes at 24 h (Figs. 1, A
and B, and 2A). The myotubes present in
thrombin-treated cultures were considerably smaller and had fewer
nuclei than those in control cultures. In cells incubated for over
24 h, thrombin was added again to counteract any loss in activity.
Examination at 32 h showed that a considerable number of cells in
both thrombin-treated and untreated populations had died. However,
untreated wells still contained a much greater proportion of nuclei in
myotubes than did the thrombin-treated wells. To determine the
concentration dependence of the thrombin effect, primary rat muscle
cultures were exposed to various doses of thrombin for 24 h. The
maximal efficacy was achieved at a concentration of 100 nM
(Fig. 2B), which is the concentration required for optimal receptor-mediated responses to thrombin in a number of cell
systems (9, 23-25).
The involvement of PAR-1 activation in thrombin-mediated effects was
examined by determining whether similar effects occurred in response to
TRAP-1. Cells were treated with TRAP-1, which was added every 4 h
over a 24-h period. Neither 50 µM nor 100 µM TRAP-1 was able to mimic the effect of thrombin on
myoblast fusion (Fig. 2C). Proteolytically inactive
PPACK-thrombin had no effect on myotube formation, showing that the
enzymatic activity of thrombin is required (Fig. 2C).
Thrombin Prevents Myoblast Death--
Cells cultured under
conditions that induced myoblast fusion (described above) were found to
undergo cell death, which was already detectable 12 h after serum
deprivation. In contrast, thrombin treatment caused a significant
inhibition of cell death (Figs. 1 and 3).
The dying cells showed the typical morphological features of
apoptotic cells, with clumping and aggregation of chromatin (Fig.
3, A and C). This phenomenon was paralleled by the occurrence of DNA strand breaks, detectable by the TUNEL reaction (Fig. 3, B and D). In many culture systems,
induction of apoptosis is dependent on de novo protein
synthesis (26). To assess whether protein synthesis was also required
for cell death in our system to occur, cells were treated with
cycloheximide (5 µM) for 30 min at the time of serum
deprivation, and then the medium was replaced with fresh serum-free
medium, and the cell viability was determined 24 h later. Cell
death in cultures treated with cycloheximide (19.5 ± 0.5%) was
significantly lower (p < 10
In time course studies, cells were treated with thrombin (100 nM) continuously from the time of serum deprivation but
were fixed and analyzed for apoptosis at various times. The thrombin effect was already detectable at 12 h. At least 50% of cells in control cultures were apoptotic at 24 h, whereas in
thrombin-treated cultures less than 15% of cells were apoptotic (Fig.
4A). At 32 h, when 75%
of cells in control cultures were apoptotic, thrombin still exerted a
significant protective effect. A further time course experiment was
carried out to determine whether the continued presence of thrombin was
required. Cells were exposed to thrombin for 0, 1, 6, 12, or 24 h
following serum deprivation, at which time medium was replaced with
fresh serum-free medium. Cells were fixed and examined at 24 h.
The results demonstrated that a 1-h treatment with thrombin at the time
of serum deprivation was sufficient to provide almost complete
protection from apoptosis (Fig. 4B). Thrombin inhibited
apoptosis over a range of concentrations, with optimal protection
occurring at 100 nM (Fig. 4C).
In the presence of catalytically inactive PPACK-thrombin, the level of
apoptosis was 88 ± 1.5% of that seen in control cultures, indicating that proteolytic activity is required for the protective effect of thrombin. Experiments were carried out to investigate whether
the inhibition of apoptosis was specific for thrombin. None of the
other serine proteases tested was able to inhibit apoptosis. In the
presence of urokinase or factor Xa (100 nM), apoptosis as a
percentage of control values was 100 ± 3.3 or 96 ± 1.0%,
respectively. Plasmin, tissue plasminogen activator, or chymotrypsin at
100 nM caused death and detachment of all cells from the substratum.
Mechanism of the Effects of Thrombin on Myoblasts--
Further
experiments were carried out to determine whether the effect of
thrombin was mediated by one of the known thrombin receptors, PAR-1,
-3, or -4. Inhibition of apoptosis by thrombin appears not to be
mediated via PAR-1 activation, because TRAP-1 did not mimic the effect
of thrombin at concentrations able to elicit
[Ca2+]i responses in such cells. In the presence
of TRAP-1 at 50 or 100 µM, apoptosis as a percentage of
control values was 93 ± 9.8 or 87 ± 5.3%, respectively.
TRAP-1 was added every 4 h over a period of 24 h to
compensate for a possible loss of the peptide activity. In one
experiment in which the aminopeptidase inhibitor, amastatin, was added
to the culture medium to rule out the possibility that TRAP-1 was being
degraded, the peptide was still unable to inhibit apoptosis. Reverse
transcriptase-polymerase chain reaction was carried out to investigate
whether myoblasts express PAR-3. Because the sequence for rat PAR-3 was
unknown, PAR-3 primers based on the mouse sequence were designed and
tested in RNA extracted from mouse cells. This approach is valid
because thrombin was found to exert the same protective effect in mouse myoblasts as in rat cells. As a positive control, PAR-3 primers detected PAR-3 transcript in mouse spleen. The same primers failed to
detect PAR-3 in mouse myoblasts. Experiments investigating the role of
PAR-4 made use of mouse TRAP-4 and mouse myoblasts because the rat
PAR-4 sequence is unknown. Cells treated with TRAP-4 (500 µM) did not exhibit [Ca2+]i
transients in response to TRAP-4, nor were they rescued from apoptosis,
undergoing 114 ± 7.9% of control levels of apoptosis. The
concentration of TRAP-4 used (500 µM) has previously been shown to activate PAR-4 in mouse cells (8).
Because the above experiments demonstrated that effects of thrombin on
myoblast apoptosis and fusion are not mediated by any of the known
thrombin receptors, further experiments were carried out to determine
whether the substrate of thrombin is a cell surface protein or a
protein secreted into the medium of myoblast cultures. When thrombin
was added to medium collected from myoblast cultures 12 or 24 h
after serum deprivation and then inactivated by the addition of PPACK,
the resulting medium had no apoptosis inhibitory activity (Table
I). Medium was also collected from cells
exposed to thrombin for 24 h after serum deprivation. Such medium
was treated with PPACK until no thrombin activity was detectable. This
medium contained inhibitory activity in apoptosis and fusion assays
almost as potent as that of thrombin (Table I). In a final experiment,
cells were treated with thrombin for 1 h following serum
deprivation and then the medium was replaced with fresh serum-free
medium without thrombin. The latter medium was harvested and used in
apoptosis and fusion assays and found to have accumulated inhibitory
activity with time (Fig. 5).
Expression patterns of PAR-1 in primary cultures of myoblasts
undergoing differentiation (16), as well as in developing muscle (17),
led us to predict that thrombin inhibits myoblast fusion. At the time
that our experiments were being undertaken, a description of the
inhibition of fusion by thrombin in the C2C12 myoblast cell line was
published (25). The demonstration in the present study that this also
occurs in primary myoblast cultures suggests that this is a
reproducible and potentially important role for thrombin in myoblast
function. Contrary to expectations, the effect of thrombin on fusion
does not appear to be mediated by PAR-1, because TRAP-1 was not able to
mimic it. It seemed possible that the TRAP-1 failed to elicit an effect
because it was degraded during the course of the experiments; it has
previously been demonstrated that an aminopeptidase is able to degrade
TRAP-1 (27). This possibility is, however, unlikely for the following
reasons: 1) in the experiments presented here, fresh TRAP-1 was added
to cultures every 4 h to ensure that adequate concentrations were
maintained throughout the culture period; 2) addition of the
aminopeptidase inhibitor, amastatin, to culture medium did not alter
the failure of cells to respond to TRAP-1; and 3) a 1-h treatment with
thrombin was sufficient to elicit a significant effect, indicating that prolonged activity of the peptide was unnecessary.
In the above-mentioned study of the effect of thrombin on C2C12 cell
fusion, thrombin was also shown to inhibit myogenin expression, an
effect that could be mimicked by TRAP-1 (25). These results, in
combination with our own showing that thrombin causes a
PAR-1-independent inhibition of fusion, suggest that thrombin regulates
myoblast differentiation through multiple pathways.
Our observation that thrombin inhibits myoblast apoptosis is novel and
of considerable interest. Vaughan et al. (28) have shown
that thrombin protects rat primary astrocytes and hippocampal neurons
from cell death induced by hypoglycaemia and oxidative stress but did
not describe the cell death inhibited by thrombin as apoptosis. In the
current study the myoblast death inhibited by thrombin has been
characterized as an apoptotic process not only on the basis of nuclear
morphology and the presence of DNA strand breaks but also because of
its requirement for protein synthesis demonstrated by the experiments
with cycloheximide. The thrombin-induced protection from cell death
observed by Vaughan et al. (28) appears to be mediated by
PAR-1, because it can be reproduced using TRAP-1. The protective effect
of thrombin in the current study uses a different pathway, because it
is not mimicked by TRAP-1.
The failure of primary myoblast cultures to express PAR-3 and their
failure to respond to TRAP-4 in terms of apoptosis, fusion, or
[Ca2+]i indicate that neither PAR-3 nor PAR-4
mediates the observed responses to thrombin. If none of the known
thrombin receptors mediates inhibition of fusion and apoptosis in
myoblasts, how does thrombin exert these effects? Both effects appear
to be dependent on the proteolytic activity of thrombin, because PPACK
thrombin was inactive in both assays. The effect is specific for
thrombin, because no other serine protease tested was able to mimic the
activity. Fig. 6 illustrates
schematically the possible pathways of thrombin activity that have been
considered in the current study. The possibility that thrombin exerts
effects on myoblasts through cleavage of proteins secreted into the
medium (Fig. 6, pathway 1) was ruled out by the
demonstration that thrombin treatment of myoblast-conditioned medium
did not generate inhibitory activity. Further information about the
mechanism of the action of thrombin was obtained by experiments
demonstrating that conditioned medium from thrombin-treated cells (in
which the thrombin had been inactivated) could substitute for thrombin
in apoptosis and fusion assays. The question arose of whether thrombin
was acting on a cellular receptor to induce secretion of an inhibitor
of apoptosis (Fig. 6, pathway 2) or simply releasing an
inhibitor of apoptosis from the cell surface or extracellular matrix
(Fig. 6, pathway 3). The fact that activity capable of
inhibiting apoptosis and fusion continues to accumulate in medium up to
20 h after the removal of thrombin indicates that thrombin is not
simply releasing activity from the cell surface or matrix. Thus we are left to conclude that the substrate of thrombin is an unidentified thrombin receptor (pathway 2).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin was prepared as described
(18). PPACK-thrombin was prepared by treating thrombin with the
inhibitor D-Phe-Pro-Arg-CH2Cl according to
Stone et al. (19) and displayed less than 0.1% of the
initial activity of the protease. Factor Xa was a generous gift from
Dr. B. Le Bonniec (INSERM, Paris). Urokinase, plasmin, chymotrypsin,
and tissue plasminogen activator were from Sigma. Rat PAR-1-activating
peptide (thrombin receptor agonist peptide; TRAP-1) was a 17-amino acid
peptide (SFFLRNPSENTFELVPL) synthesized by Dr. P. Thompson (Department
of Biochemistry and Molecular Biology, Monash University). Mouse
PAR-4-activating peptide (TRAP-4) was a 6-mer (GYPGKF) synthesized by
Dr. H. Keah (Department of Biochemistry and Molecular Biology, Monash
University). Both peptides were purified by high pressure liquid
chromatography, and their compositions were verified by amino acid
analysis. Polymerase chain reaction primers were obtained from Pacific
Oligos (Lismore, Australia). Cell culture media were from Life
Technologies, Inc., and other reagents were from Sigma unless otherwise stated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (157K):
[in a new window]
Fig. 1.
Effect of thrombin on myoblast fusion and
survival. Subconfluent myoblast-enriched cultures were deprived of
serum then cultured for 24 h in the presence of thrombin (100 nM; A and B) or without additives
(C and D). A and C,
cultures stained with DAPI; B and D, the
corresponding fields stained for the presence of desmin. The
large arrows indicate myotubes, and the small
arrows indicate some of the nuclei detectable as undergoing
apoptosis. Bar, 50 µm.
View larger version (27K):
[in a new window]
Fig. 2.
Myoblast fusion is inhibited in the presence
of thrombin. A, subconfluent myoblast-enriched cultures
were incubated in serum-free medium in the presence or absence of
thrombin (100 nM) for the times indicated. Cultures were
fixed, stained for the presence of desmin, and then counterstained with
DAPI. Nuclei in myotubes were counted as a proportion of total nuclei
in desmin-positive cells; results are presented as the means ± S.E. B, subconfluent myoblast-enriched cultures were
incubated for 24 h in serum-free medium containing thrombin at the
concentrations indicated. Cultures were processed and nuclei counted as
above. C, subconfluent myoblast-enriched cultures were
incubated for 24 h in serum-free medium without additives or in
the presence of thrombin, TRAP-1 or PPACK-thrombin. Cultures were
processed and nuclei counted as above.
4) than in
control cultures (41.7 ± 1.1%).
View larger version (114K):
[in a new window]
Fig. 3.
Effect of thrombin on myoblast survival.
Subconfluent myoblast-enriched cultures were incubated for 24 h in
serum-free medium containing thrombin (100 nM; A
and B) or without additives (C and D).
A and C, cultures stained with DAPI. B
and D, the corresponding fields stained with the TUNEL
reagents. Arrows indicate individual apoptotic myoblasts
detectable both by DAPI and TUNEL staining. Bar, 50 µm.
View larger version (22K):
[in a new window]
Fig. 4.
Myoblast apoptosis is delayed in the presence
of thrombin. A, subconfluent myoblast-enriched cultures were
incubated in serum-free medium in the presence or absence of thrombin
(100 nM) for the times indicated when cultures were fixed,
stained for the presence of desmin, and counterstained with DAPI.
Apoptotic myoblast nuclei were counted as a proportion of total nuclei
in desmin-positive cells; results are presented as the means ± S.E. B, subconfluent myoblast-enriched cultures were
incubated in serum-free medium in the presence or absence of thrombin
(100 nM) for the times indicated, when medium was replaced
with fresh serum-free medium. At 24 h cultures were processed and
nuclei counted as above. C, subconfluent myoblast-enriched
cultures were incubated for 24 h in serum-free medium containing
thrombin at the concentrations indicated. Cultures were processed and
nuclei counted as above.
Investigations into the mechanism of the effect of thrombin on myoblast
apoptosis and fusion
View larger version (36K):
[in a new window]
Fig. 5.
Detection of a mediator of the effects of
thrombin in medium conditioned by thrombin-treated myoblasts.
A, apoptosis. Subconfluent myoblast-enriched cultures were
incubated for 24 h in serum-free medium containing thrombin or no
additives or in serum-free myoblast-conditioned medium (CM).
Conditioned medium had been harvested at various times (as indicated)
after a 1-h exposure of cells to thrombin (as described under
"Materials and Methods"). Cultures were fixed, stained for the
presence of desmin, and then counterstained with DAPI. Apoptotic
myoblast nuclei were counted as a proportion of total nuclei in
desmin-positive cells; results are presented as the means ± S.E.
B, fusion. Subconfluent myoblast-enriched cultures were
treated, fixed, and stained as above. Nuclei in myotubes were counted
as a proportion of total nuclei in desmin-positive cells; results are
presented as the means ± S.E.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (35K):
[in a new window]
Fig. 6.
Schematic representation of possible pathways
for the inhibitory effects of thrombin on myoblast apoptosis and
fusion. Pathway 1, cleavage of a protein secreted into
the medium by myoblasts, resulting in generation of an inhibitor of
apoptosis and fusion (IAF). Pathway 2, cleavage
and activation of a cell surface receptor resulting in synthesis and
secretion of a downstream inhibitor of apoptosis and fusion.
Pathway 3, cleavage and release of a cell surface- or
extracellular matrix-bound inhibitor of apoptosis and fusion. The
results presented here indicate that pathway 2 is the major pathway
involved in inhibition of apoptosis and fusion of thrombin.
The results presented here suggest that thrombin plays an important role in muscle development by affecting the functional state of myoblasts in several different ways. Thrombin has previously been shown to increase myoblast number (16), and our results indicate that thrombin inhibits both myoblast fusion and apoptosis. It is likely that during normal muscle development and growth an equilibrium between thrombin and other growth and/or differentiation factors determines the number of cells that progress through the full sequence of differentiation culminating in myotube formation. The question arises of whether thrombin is present in developing muscle in the absence of vascular disruption. Recent studies investigating the expression of prothrombin in developing muscle confirm that the protein is present in this tissue (14). In addition, thrombin activity is increased in myotube cultures after cholinergic stimulation, in part by increasing prothrombin transcription (15). Our results suggest that thrombin produced by myotubes during muscle development would take part in a negative feedback loop, limiting the number of myoblasts that fuse, and ensuring that some cells capable of proliferation remain to meet future needs.
In normal adult muscle apoptosis appears to be a rare event (29), and
our results suggest that thrombin may be important in maintaining this
situation. It is also likely that thrombin plays a role in the recovery
from muscle damage involving vascular disruption, partly through its
effect on myoblast survival. Indeed, a thrombin concentration of 360 nM has been measured in the fluid phase of clotted blood
(30), well above the levels required for maximal responses in our
culture system. In a number of pathological situations, including
exercise-induced muscle damage and muscular dystrophy, the proportion
of muscle cells undergoing apoptosis increases (29, 31, 32). It is of
considerable interest to note that the incidence of apoptosis is
elevated in skeletal muscle subjected to disuse or denervation, as
compared with normal muscle (33, 34). Because cholinergic stimulation
of muscle fibers results in an increase in thrombin activity (15), it
is tempting to speculate that a decrease in thrombin activity in
denervated muscle allows the resultant apoptosis to occur.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. J. Bower, Dr. L. Austin, Dr. R. Pike, and Dr. H. Suidan for helpful advice.
![]() |
FOOTNOTES |
---|
* This work was supported by Australian Research Council Project Grant A09702061 and National Health and Medical Research Council Project Grants 970498 and 970490 (to the late Prof. S. R. Stone).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. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed. Tel.:
61-3-9344-7360; Fax: 61-3-9344-7374; E-mail:
e.mackie{at}vet.unimelb.edu.au.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PAR, protease-activated receptor; PPACK, D-Phe-Pro-Arg-CH2Cl; TRAP, thrombin receptor activating peptide; DAPI, 4',6'-diamidino-2-phenylindole; TUNEL, TdT-mediated dUTP nick end labeling; PBS, phosphate-buffered saline.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|