(Received for publication, February 12, 1997, and in revised form, May 12, 1997)
From the Dipartimento di Ricerca Biologica e
Tecnologica (DIBIT), San Raffaele Scientific Institute, via Olgettina
58, 20132 Milan, Italy and ¶ Institute of Histology, School of
Medicine, University of Siena, 53100 Siena, Italy
In vertebrate skeletal muscles, the type 1 isoform of ryanodine receptor (RyR1) is essential in triggering
contraction by releasing Ca2+ from the sarcoplasmic
reticulum in response to plasma membrane depolarisation. Recently, the
presence of another RyR isoform, RyR3, has been detected in mammalian
skeletal muscle cells, raising the question of the eventual relevance
of RyR3 for muscle cell physiology. The expression of RyR3 was
investigated during differentiation of skeletal muscle cells. Using
antibodies able to distinguish the different RyR isoforms and Western
blot analysis, the RyR3 protein was detected in the microsomal
fractions of differentiated skeletal muscle cells but not of
undifferentiated cells. Accordingly, blocking muscle differentiation by
the addition of either transforming growth factor- or basic
fibroblast growth factor prevented the expression of the RyR3 protein.
In differentiated skeletal muscle cells, RyR3 was expressed independent
of cell fusion and myotube formation. The expression of RyR3 was also
investigated during development of the diaphragm muscle. The RyR3
content in the diaphragm muscle increased between the late stage of
fetal development and the first postnatal days. However, at variance
with RyR1, which reached maximum levels of expression 2-3 weeks after
birth, the expression of RyR3 was found to be higher in the neonatal
phase of the diaphragm muscle development (2-15 days after birth) than in the same muscle from adult mice. The differential content of RyR3 in
adult skeletal muscles was found not to be mediated by neurotrophic
factors or electrical activity. These findings indicate that RyR3 is
preferentially expressed in differentiated skeletal muscle cells. In
addition, during skeletal muscle development, its expression is
regulated differently from that of RyR1.
Ryanodine receptors, together with the inositol trisphosphate (InsP3) receptors,1 form a superfamily of intracellular channels that mediate calcium release from intracellular calcium stores into the cytosol (1-5). Three isoforms of RyRs, encoded by three different genes, have been identified. RyR1 and RyR2 are predominantly expressed in skeletal and cardiac muscles (6-9), respectively, and at lower levels in other tissues (10). A third isoform named RyR3 has been found to be present at low levels in almost all tissues (11-13), but so far no convincing evidence for a preferential association with a specific cell type or function has been obtained.
Most of our knowledge on RyR1 and RyR2 isoforms of the calcium release channel comes from studies in vertebrate striated muscles, where they play an important role for the release of calcium from the sarcoplasmic reticulum to activate contraction (14-15). Transduction of the action potential signal from the cell surface to the interior of muscle cells, often referred to as excitation-contraction coupling, involves the dihydropyridine receptors (DHPRs) on the plasma membrane and RyRs on the sarcoplasmic reticulum (16-17). Despite some similarities, the mechanism of excitation-contraction coupling differs in skeletal and cardiac striated muscles. In cardiac fibers, a calcium influx from the extracellular environment through the cardiac-specific DHPR activates the RyR2 isoform, probably via a calcium-induced calcium release mechanism (18-20). In adult skeletal muscle, an influx of extracellular calcium through the skeletal DHPR is apparently not required, so that it has been proposed that the skeletal muscle DHPR detects the action potential and activates the RyR1 located on the terminal cisternae of the sarcoplasmic reticulum, probably by a direct physical coupling (21-22). Accordingly, in vertebrate skeletal muscles, DHPRs and RyRs are colocalized in clusters soon after their synthesis and form the junctional domains observed in the space between transverse tubules (T tubules) and the terminal cisternae of the sarcoplasmic reticulum (23). Although junctional domains start to organize during the fetal stages of development, it is only after birth that this system undergoes a final reorganization (24). In mammals, within the period of time required after birth for the development and organization of a mature system of junctional domains, some quantitative and qualitative changes also occur in the biochemical composition of this system. In a few days after birth, the expression of DHPR genes is strongly increased, attaining maximal levels before the gene encoding the RyR1 isoform, which requires about 3-4 weeks to reach the high levels that are maintained throughout adult life (25). During the postnatal period of skeletal muscle development, the transient expression of the cardiac isoform of DHPR has also been observed (26-27). The immature state of the excitation-contraction coupling apparatus at birth results in the apparent requirement of extracellular calcium in order for electrical stimulation to trigger contraction (28-30). In mammalian skeletal muscles, the organization of a mature excitation-contraction coupling apparatus is established within 4 weeks of birth (24).
With respect to the biochemical composition of the
excitation-contraction coupling apparatus, it has been shown that in
most avian, amphibian, and fish skeletal muscles, two isoforms of RyRs named and
, which correspond to mammalian RyR1 and RyR3
(31-35), are expressed. Recent evidence has indicated that also in
mammalian skeletal muscles, in addition to RyR1, the RyR3 isoform is
present (10, 36-37). In mammalian skeletal muscles, RyR3 is expressed at lower levels than RyR1; however, whereas RyR1 expression appears to
be rather homogeneous, it is intriguing to note that the levels of RyR3
vary among different skeletal muscles, with maximal expression in the
diaphragm and intermediate levels in the soleus. In other skeletal
muscles, RyR3 levels are either extremely low or below detection limits
(36). To further extend our knowledge of RyR3 in mammalian skeletal
muscles, we analyzed whether the expression of RyR3 was associated with
the differentiated state of cultured myocytes and studied its
expression during the development of the diaphragm muscle. RyR3 content
was also analyzed in denervated adult muscles to verify the possible
influence of neurotrophic factors and electrical activity on the
differential expression of the RyR3 isoform among different skeletal
muscles.
Mouse C2C12 cells (38)
were grown at 37 °C in a humidified atmosphere of 5%
CO2 in Dulbecco's modified Eagle's medium supplemented with 2 mM glutamine (BioWittaker, Inc.), 100 µg/ml
streptomycin, 100 units/ml penicillin (BioWittaker), 1 mM
sodium pyruvate (Bio-Wittaker), 10% heat-inactivated fetal calf serum
(Life Technologies, Inc.). The cells were subcultured at low density in
10-cm-diameter Petri dishes every 3 days. To induce differentiation,
cells were plated on gelatin-coated plates at 2 × 105
cells/cm2 in Dulbecco's modified Eagle's medium 10%
fetal calf serum. After 4 h, the medium was changed to a
differentiation medium consisting of Dulbecco's modified Eagle's
medium supplemented with 10 µg/ml insulin (Sigma) and 5 µg/ml
apotransferrin (Sigma). In experiments with growth factors and EGTA,
the differentiation medium was supplemented with either 5 ng/ml
transforming growth factor (TGF-
), 90 ng/ml basic fibroblast
growth factor (bFGF), or 1.4 mM EGTA (39-41). Cells were
harvested at the indicated times.
Mouse diaphragm muscles
were isolated from mice of 18 days of fetal development at days 2, 5, and 15 after birth and from adult mice (>2 months old). Bovine tissues
were used to prepare the microsomal fractions of hippocampus, skeletal,
and cardiac muscles used as controls. Microsomes were prepared as
described previously (10, 36). Briefly, tissue samples or cultured
cells were homogenized in ice-cold buffer A (320 mM
sucrose, 5 mM Na-Hepes, pH 7.4, and 0.1 mM
phenylmethylsulfonyl fluoride) using a Teflon potter for cells or a
Dounce homogenizer for tissues. Homogenates were centrifuged at
7000 × g for 5 min at 4 °C. The supernatant obtained was centrifuged at 100,000 × g for 1 h
at 4 °C. The microsomes were resuspended in buffer A and stored at
80 °C. Protein concentration of the microsomal fraction was
quantified using the Bradford protein assay kit (Bio-Rad).
Microsomal proteins were separated by SDS-polyacrylamide gel electrophoresis as described (10). Proteins were then transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc.) using a transfer buffer containing 192 mM glycine, 25 mM Tris, 0.01% SDS, and 10% methanol for 5 h at 350 mA at 4 °C. Filters were blocked for 3 h in a buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 0.2% Tween 20, 5% no-fat milk and incubated overnight at room temperature with specific antibodies. Polyclonal rabbit antisera were developed against purified glutathione S-transferase fusion proteins corresponding to the region of low homology situated between the transmembrane domains 4 and 5 (divergent region 1) of the RyR1, RyR2, and RyR3 proteins as described previously (10). These antibodies are able to distinguish the three RyRs and have been shown not to cross-react with each other (10, 32, 36). A monoclonal antibody against Ca2+-ATPase (YIF4) was kindly provided by Dr J. M. East. The antibodies against the RyRs were used at 1:3000 dilution, whereas the cell culture supernatant containing the YIF4 monoclonal antibody was used undiluted. Antigen detection was performed using the alkaline phosphatase detection method.
Calcium MeasurementsUndifferentiated and differentiated
C2C12 cells were detached from plates with a
trypsin/EDTA mix and washed with fresh cell culture medium by
centrifugation at low speed for 5 min. Cells were then resuspended in
Krebs-Ringer-Hepes medium (125 mM NaCl, 5 mM
KCl, 1.2 mM KH2PO4 , 1.2 mM MgSO4, 2 mM CaCl2 , 6 mM glucose, and 25 mM Hepes adjusted to pH
7.4 with NaOH) at a concentration of 5 × 106
cells/ml. Fura 2-AM (Calbiochem) in anhydrous dimethyl sulfoxide was
added to a final concentration of 5 µM, and the cells
were incubated for 30 min at room temperature in the dark. The cells were then diluted to a final concentration of 106 cells/ml.
Before use, 750-µl aliquots of cells were washed and resuspended in
Krebs-Ringer-Hepes medium with 200 µM sulfinpyrazone (Sigma) and transferred to a cuvette equipped with a magnetic stirrer
for whole-cell population calcium measurement. The Fura 2 fluorescence
was recorded on a luminescence spectrometer LS 50 (Perkin-Elmer) with
excitation at 345 nm and emission at 490 nm. Maximum and minimum
fluorescence (Fmax and
Fmin) were determined by the addition of 5 mM EGTA in Tris base, 40 mM Tris, 0.1% Triton X-100 (Fmin), and 3 mM
CaCl2 (Fmax). [Calcium]i
was calculated from the fluorescence Fmax and
Fmin using a dissociation constant for the Fura
2·calcium complex of 225 nM and the equation [calcium] = [(F Fmin)/(Fmax
F)] × Kd.
In skeletal muscle cells, calcium release through RyRs
is associated with the expression of the differentiated phenotype and the regulation of muscle contraction, whereas the pathway that operates
through the InsP3 receptors appears to be independent of
differentiation (42-43). The organization of calcium stores and the
expression of calcium release channels has been extensively studied
using in vitro cultured cell lines and in developing and adult skeletal muscles (39, 42, 44-46). To define the calcium stores
present in proliferating and differentiated muscle cells, we performed
fluorometric analysis of cytoplasmic calcium concentration using Fura 2 dye and the C2C12 cell line as an in
vitro model system of muscle differentiation and myotube
development. Cells were stimulated with either bradykinin, a specific
agonist of the InsP3 receptor family of intracellular
calcium channels and with caffeine, a specific agonist of RyRs. As
shown in Fig. 1, stimulation with 100 nM bradykinin induced a transient increase of intracellular
calcium concentration in both differentiated and undifferentiated
C2C12 cells (Fig. 1, lower panels).
Instead, undifferentiated cells (Fig. 1, left upper panel)
did not show any caffeine-induced calcium release, whereas a transient
increase in intracellular calcium concentration after 20 mM
caffeine stimulation was observed only in 4 day-differentiated cells
(Fig. 1, right upper panel).
RyR3 Expression in Mammalian Skeletal Muscle Cells
A second
isoform of calcium release channel, RyR3, has been recently detected in
some mammalian skeletal muscles (36). To further extend this analysis,
we measured the expression of the RyR3 isoform in microsomal fractions
prepared from undifferentiated and fully differentiated
C2C12 cells. Western blot analysis of the
microsomal proteins using antibodies able to distinguish the three RyR
isoforms is shown in Fig. 2. The three
proteins recognized by these antisera run in SDS gels with slightly
different relative mobilities, with RyR1 showing the higher molecular
weight and RyR3 showing the smaller relative mobility. The specificity
of the antisera used is confirmed by isoform-specific signal displayed against skeletal muscle, cardiac muscle, and hippocampal microsomal preparations used as controls (see figure legends). Both RyR1- and
RyR3-specific bands are detected in differentiated culture (lanes
2 and 12), whereas they cannot be detected in
undifferentiated growing cells (lanes 1 and 11).
RyR2 protein, as expected, was not detected in either preparation
(lanes 6 and 7). Similar results were obtained
with microsomal fractions prepared from differentiated and
undifferentiated murine BC3H1 cells and rat L6 cells (data not shown).
Although at this level we cannot distinguish the calcium released
through RyR3 from the calcium released through the RyR1 channels, these
results indicate that the presence of functional calcium release stores
operated by caffeine (i.e. ryanodine receptors) parallels
the regulated expression of both RyR isoforms of calcium release
channels.
The Expression of RyR3 in Mammalian Skeletal Muscle Cells Is Dependent on Differentiation
TGF- and bFGF are known to
interfere with myogenic differentiation without inducing a significant
effect on cell proliferation (39, 45, 47). They can be used, therefore,
to verify whether RyR3 expression is related to the activation of the
skeletal muscle differentiation program or is a consequence of the
withdrawal from the cell cycle due to the specific culture conditions
used for the induction of differentiation. Fig.
3 shows the results obtained from Western
blot analysis of C2C12 cells induced to differentiate in serum-free medium and in serum-free medium
supplemented with TGF-
and bFGF for 2 and 4 days. The data reported
in Fig. 3 reveal a significant reduction of the RyR1 and
Ca2+-ATPase levels both at 2 and 4 days of treatment when
compared with normally differentiated cells. Analysis of the RyR3
content under these conditions revealed that RyR3, undetectable in
undifferentiated growing cells, is clearly expressed at day 2 of
culture in serum-free medium, indicating that this channel is expressed
in the early stages of differentiation and further increases at day 4 of differentiation. RyR3 signal is significantly reduced in microsomal
membranes of cells cultured in serum-free medium supplemented with
either TGF-
or bFGF, suggesting that the arrest of growth is not
sufficient to induce RyR3 synthesis but that the cells have to enter
the differentiation program to express this protein.
Expression of RyR3 in Mammalian Skeletal Muscle Cells Is Not Dependent on Cell Fusion
An important step in skeletal muscle
differentiation is represented by myotube formation. Soon after plating
C2C12 in serum-free medium, myotubes and
polynucleated syncitia due to cell fusion appear. The number of
myotubes in culture rises dramatically between days 4 and 5. Extracellular calcium is known to be necessary for the cell fusion
process leading to myotubes development. To analyze whether RyR3
expression in differentiated muscle cells was dependent on the process
of cell fusion, C2C12 cells were induced to
differentiate with 1.4 mM EGTA to prevent cell fusion and
myotube formation. This protocol is known not to affect the execution
of the differentiation program at the biochemical level
(i.e. expression of muscle-specific proteins is not
prevented) (40-41). As expected, the fraction of polynucleated cells
at day 4 in EGTA-supplemented medium was about 5% that observed in
control differentiating cultures. However, despite a remarkable effect
of EGTA on the morphological differentiation, muscle-specific proteins
(see Ca2+-ATPase and RyR1 content in Fig.
4) are present at levels comparable with
normally differentiated control cells, indicating that indeed the cells
underwent biochemical differentiation although they remained
mononucleated. RyR3 was expressed in mononucleated differentiated C2C12 at levels similar to those of
polynucleated differentiated cells (Fig. 4), indicating that RyR3
expression is dependent on differentiation and is induced before and
independently of cell fusion, similar to what was observed for RyR1 and
other skeletal markers (39).
Expression of the RyR3 Isoform in Developing Skeletal Muscle
Although most muscle-specific proteins start to be
expressed during embryo development, a significant burst in the
synthesis of these proteins occurs around birth. With respect to the
components of the excitation-contraction coupling apparatus, it has
been shown that the patterns of expression of RyR1 and DHPR
1 subunit are distinct after birth, with the DHPR
reaching maximal levels of expression earlier than RyR1 (25). In this
context, it was interesting to investigate the in vivo
developmental pattern of expression of the RyR3 protein. The diaphragm
muscle was chosen in view of its relatively high content in RyR3
protein (36). Muscle tissue was isolated from mice at day 18 of fetal
development (F18), at day 2 (P2), day 5 (P5), and day 15 (P15) after
birth and from adult mice (i.e. 60-day-old mice). Both RyR1
and RyR3 were detected at the earliest developmental stage analyzed, as shown in Fig. 5. A relatively constant
increase in RyR1 and Ca2+-ATPase can be observed in
microsomes prepared from different stages, in agreement with published
data (46). With respect to the RyR3 content, a significant increase was
observed in the diaphragm muscle of mice from stages F18 (Fig. 5,
lane 1) to P2 (not shown), P5 (Fig. 5, lane 2),
and P15 (not shown). Surprisingly, a decrease in the RyR3 content was
detected in diaphragm muscles prepared from adult mice (Fig. 5,
lane 3). The same results were obtained in three independent
experiments, ensuring that the observed differences in the levels of
RyR3 in diaphragm muscles at the different days analyzed were
significant. Thus, we conclude that RyR3 is expressed at higher levels
in neonatal than in adult diaphragm muscle.
Expression of RyR1 and RyR3 in Adult Skeletal Muscles Is Not Dependent on Electrical Activity/Neurotrophic Factors
Expression
of many skeletal muscle genes and proteins has been shown to be
dependent on various factors including nerve-induced electrical
activity and neurotrophic factors (48-49). In light of the observed
differences in the RyR3 isoform content among mammalian skeletal
muscles, we analyzed last whether the expression of RyR3 was regulated
by innervation. This was analyzed in rat soleus and extensor digitorum
longus muscles after removal of a section of the sciatic nerve and in
the right hemidiaphragm muscle after unilateral removal of a section of
the phrenic nerve, using an intercostal approach. Microsomes were
isolated from the indicated muscles 10 days after nerve recision and
from the corresponding contralateral control muscles. Proteins were
size-fractionated on SDS-polyacrylamide gel electrophoresis,
transferred to nitrocellulose membranes, and decorated with antibodies
able to discriminate the RyR1, RyR2, and RyR3 isoforms. Results
reported in Fig. 6 indicate that the
levels of RyR1 and RyR3 in denervated muscles (lanes 2, 4, and 6, respectively) were essentially unchanged with respect to controls (lanes 1, 3, and 5,
respectively), thus indicating that expression of RyR3 in soleus and
diaphragm as well as its absence in the extensor digitorum longus
muscle is independent of muscle innervation. No expression of RyR2 in
denervated muscles was detected.
The process of skeletal muscle development is a complex mechanism that implies a fine regulation of the expression of contractile and sarcoplasmic proteins. This has been extensively studied using both primary myoblasts and cell lines able to differentiate and form myotubes in vitro (24). Studies on the structural and functional organization of intracellular calcium stores indicated that the activity of these organelles depends on a complex level of organization, including the regulated expression of the genes encoding proteins that transport calcium ions in the luminal side of these vesicles, calcium binding proteins, and calcium release channels (1). Previous studies on the composition and function of intracellular calcium stores during in vitro differentiation of skeletal muscle cells revealed that, whereas InsP3-sensitive stores can be present in undifferentiated and in differentiated cells, caffeine-sensitive stores can be detected only in differentiated cells (42-43). These observations suggest that the InsP3 receptor plays a role in regulating intracellular calcium homeostasis with respect to housekeeping activities, whereas expression and function of the skeletal muscle isoform, RyR1, have been shown to be mainly related to the functions of the differentiated muscle cells.
In this report we have analyzed the expression of the RyR3 isoform
using cultured myogenic cell lines and during the development of
skeletal diaphragm muscle. Western blot analysis of microsomal fractions prepared both from undifferentiated and differentiated cells
indicated that two isoforms of calcium release channel, RyR1 and RyR3,
were present in differentiated muscle cells but not in growing,
undifferentiated cells. This was confirmed in experiments where RyR3
expression was completely abolished when differentiating cells were
cultured in the presence of growth factors such as bFGF and TGF-,
which inhibit myogenic differentiation. This indicates that as reported
for RyR1 and other muscle proteins (39, 45, 47), RyR3 synthesis is not
dependent on cell cycle withdrawal but is rather related to the
activation of the myogenic differentiation program. In addition, RyR3
expression was found to occur before and independent from the
appearance of morphologically differentiated myotubes, as stated by
experiments where cell fusion and myotube formation was prevented by
removal of extracellular calcium with EGTA. In conclusion, all of the
experiments performed with differentiating myoblasts indicate that
expression of the RyR3 isoform in muscle cells shows a trend similar to
that of most of the known muscle-specific proteins.
In nonmammalian skeletal muscles, the expression of equivalent levels
of two isoforms of calcium release channels, namely and
, is a
predominant feature of most species. The two channels display distinct
calcium release properties (50-52), suggesting that RyR1 and RyR3
isoforms may properly fit with the functional model based on
morphological studies of triads at the electron microscope, where one
out of every two RyRs is not opposed to DHPRs (53-54). How the
uncoupled RyRs are activated is yet unclear, although their activation
could be mediated by the isoforms coupled to the DHPRs (14). Up to now,
there is no direct evidence for a role of the RyR3 isoform in mammalian
skeletal muscle physiology and excitation-contraction coupling. Lack of
response to electrical stimulation has been reported in the skeletal
muscle of mice and chickens that carry mutations which abolish the
expression of the RyR1/
-RyR isoform, indicating that RyR3 cannot
substitute for RyR1 function in this process. Caffeine stimulation
does, however, cause calcium release in the RyR1-deficient mouse
myocytes in support of a potential role of RyR3 in skeletal muscle
intracellular calcium regulation, probably through a calcium-induced
calcium release mechanism (37, 55-56). The recent studies of knockout mice that do not express a functional RyR3 protein indicate that these
mice can develop normal skeletal muscle tissue in the absence of the
RyR3 isoform (57).2 However,
the presence of subtle qualitative modifications in the
excitation-contraction coupling of muscles from these mice cannot yet
be completely ruled out and requires further studies.2
Expression of most muscle-specific proteins is strongly increased after birth probably as a consequence of the activation of muscle activity and reaches maximal levels a few weeks after birth (24-25). However, the kinetics of expression of skeletal muscle genes around birth are not synchronous. Analysis of RyR3 expression during mouse skeletal muscle development revealed that RyR3 was already expressed during fetal development (F18) similar to the RyR1 isoform and that expression of both isoforms increased at birth. However, after birth, a distinct pattern of expression was observed for the two receptors. Whereas the RyR1 content continued to increase in the period from P2-P15 to adult life, the RyR3 content was found to be higher in the days following birth (P2-P15) than in the diaphragm muscle of adult mice, indicating that in this muscle RyR3 is preferentially expressed during the neonatal phase of development. This finding indicates that the relative ratio between RyR1 and RyR3 isoforms of calcium release channels in mammalian skeletal muscles differs not only among different muscles but also in the same muscle during postnatal development.
It is interesting to note that for a period of a few weeks after birth the excitation-contraction coupling apparatus of the skeletal muscle appears to be functionally immature compared with that of adult skeletal muscle. In fact, during this period skeletal muscle contraction appears to depend on external calcium (28-30). Another biochemical marker of the differences in the composition of the excitation-contraction coupling apparatus during the neonatal period of muscle development is represented by the temporary expression of the cardiac DHPR channel, which is otherwise never detected in skeletal muscles with the exception of regenerating muscles (26-27, 58-59). Therefore, although RyR3 is apparently not essential for the development of skeletal muscles, RyR3 is expressed in differentiated skeletal muscle cells, and its expression is developmentally regulated. The coexpression of the RyR1 and RyR3 calcium release channels in skeletal muscles may result in a diversification of the excitation-contraction coupling apparatus to obtain a fine regulation of muscle contraction. In nonmammalian vertebrates, differential usage of either one or two isoforms of RyRs seems to reflect the contractile properties of specific muscles (60). Thus it is not unreasonable that the different content in RyR3 observed in mammalian skeletal muscles or in at least a specific subset of them may fulfill a similar task (36). The preferential expression of RyR3 in neonatal diaphragm muscle suggests a possible role of the RyR3 isoform in excitation-contraction coupling of immature diaphragm muscle and may help to unravel the role of RyR3 in this muscle. Further work is clearly required to address this specific aspect.
We thank Emilio Clementi for helping us to set up the calcium release experiments, Stefano Schiaffino for suggestions and comments, and Larry Wrabetz for reading the manuscript.