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Am J Physiol Cell Physiol
280: C146–C154, 2001.
Expression of the Na⫹/Ca2⫹ exchanger in skeletal muscle
BODVAËL FRAYSSE,* THIERRY ROUAUD,* MARIE MILLOUR, JOSIANE FONTAINE-PÉRUS,
MARIE-FRANCE GARDAHAUT, AND DMITRI O. LEVITSKY
Faculté des Sciences et des Techniques, Université de Nantes, Nantes Cedex 3, France
Received 6 July 1999; accepted in final form 17 July 2000
sodium/calcium exchanger isoforms; gene expression
THE SODIUM/CALCIUM EXCHANGER
is a transmembrane protein, localized in plasma membrane, which ensures the
electrogenic countertransport of 3 Na⫹ against 1 Ca2⫹.
The direction of the exchange is determined by the
electrochemical transmembrane gradients of Na⫹ and
Ca2⫹ (for review, see Ref. 3). Three isoforms of the
Na⫹/Ca2⫹ exchanger coded by independent genes have
been described (38). The first, NCX1, is widely distributed in most animal cells (19, 38). The importance of its
role is well-documented in cardiomyocytes in which
Na⫹/Ca2⫹ exchange represents the major mechanism
of Ca2⫹ extrusion from the cytoplasm during diastole
(39). It has also been shown that Ca2⫹ entry via both
L-type Ca2⫹ channels and reverse Na⫹/Ca2⫹ exchange
can trigger Ca2⫹ release from the sarcoplasmic reticulum (SR) and thus activate heart cell contraction (18,
42).
* B. Fraysse and T. Rouaud contributed equally to this work.
Address for reprint requests and other correspondence: D. O.
Levitsky, CNRS EP 1593, Faculté des Sciences et des Techniques,
Université de Nantes, 2, rue de la Houssinière, Nantes Cedex 3,
France (E-mail: [email protected]).
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Increased levels of NCX1 mRNA expression and
high-Na⫹/Ca2⫹ exchange activities have also been
found in membrane vesicles obtained from brain and
kidney. In neurons, Na⫹/Ca2⫹ exchange is thought to
lower the level of intracellular Ca2⫹ after neurotransmitter release (3). The Na⫹/Ca2⫹ exchange of epithelial
cells of the distal nephron is involved in active reabsorption of Ca2⫹ (26). The specific functions of the
Na⫹/Ca2⫹ exchanger of other cell types are still undetermined. This plasma membrane protein may have a
universal function as a major pathway for extrusion of
Ca2⫹ into extracellular space, thus protecting cells
from Ca2⫹ overload (3).
It has long been considered that the contractile activity of skeletal muscle, unlike that of the heart, does
not depend on extracellular Ca2⫹ (for a review, see Ref.
30). In particular, single twitches of adult skeletal
muscle fibers are not inhibited by brief removal of
extracellular Ca2⫹ (1). Conversely, several observations have suggested that extracellular Ca2⫹ plays a
greater role in the contractile activity of developing
skeletal muscles, which do not possess a fully achieved
SR network (40), than in mature muscles. Thus the
twitch responses of a fast-twitch skeletal muscle in
2-wk-old mice are already affected by a slight lowering
of extracellular Ca2⫹ concentration and are completely
abolished in Ca2⫹-free solutions (8). Many reports have
also indicated that Ca2⫹ fluxes through a Na⫹/Ca2⫹
exchange system may contribute to the contractile activity of adult skeletal muscles (13, 20, 44, 45). These
results were usually obtained when the extracellular
medium, normally high in Na⫹, was switched to a
low-Na⫹ solution. Under these conditions, the Na⫹/
Ca2⫹ exchanger presumably operates in a “reverse
mode,” transporting Ca2⫹ into the muscle cell. Moreover, immunofluorescence data indicate that NCX1 is
present at the cell surface membrane of rat diaphragm
(14), and an outward Na⫹/Ca2⫹ exchange current has
been demonstrated in excised patches of sarcolemma
from mouse skeletal muscle (11). More recent findings
demonstrating the expression in skeletal muscle of two
other isoforms, NCX2 and NCX3 (24, 32, 38), have
raised questions about the functional role of Na⫹/Ca2⫹
exchange in different muscles. In view of the structural
and functional complexity of muscles involving the
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0363-6143/01 $5.00 Copyright © 2001 the American Physiological Society
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Fraysse, Bodvaël, Thierry Rouaud, Marie Millour,
Josiane Fontaine-Pérus, Marie-France Gardahaut,
and Dmitri O. Levitsky. Expression of the Na⫹/Ca2⫹ exchanger in skeletal muscle. Am J Physiol Cell Physiol 280:
C146–C154, 2001.—The expression of the Na⫹/Ca2⫹ exchanger was studied in differentiating muscle fibers in rats.
NCX1 and NCX3 isoform (Na⫹/Ca2⫹ exchanger isoform) expression was found to be developmentally regulated. NCX1
mRNA and protein levels peaked shortly after birth. Conversely, NCX3 isoform expression was very low in muscles of
newborn rats but increased dramatically during the first 2
wk of postnatal life. Immunocytochemical analysis showed
that NCX1 was uniformly distributed along the sarcolemmal
membrane of undifferentiated rat muscle fibers but formed
clusters in T-tubular membranes and sarcolemma of adult
muscle. NCX3 appeared to be more uniformly distributed
along the sarcolemma and inside myoplasm. In the adult,
NCX1 was predominantly expressed in oxidative (type 1 and
2A) fibers of both slow- and fast-twitch muscles, whereas
NCX3 was highly expressed in fast glycolytic (2B) fibers.
NCX2 was expressed in rat brain but not in skeletal muscle.
Developmental changes in NCX1 and NCX3 as well as the
distribution of these isoforms at the cellular level and in
different fiber types suggest that they may have different
physiological roles.
NA⫹/CA2⫹ EXCHANGER DURING MUSCLE DEVELOPMENT
then progressively decreased, whereas those of NCX3
were very low in skeletal muscle of newborn rats.
During the first postnatal weeks, the expression of
NCX3 increased dramatically. No NCX2 signals were
detected in skeletal muscle at protein or mRNA levels.
EXPERIMENTAL PROCEDURES
Animals. Male and female rats born to pregnant 3-mo-old
Wistar-Kyoto (WKY) dams were used in this study. The rats
were obtained from the R. Janvier Breeding Center and
housed in the vivarium of the Faculty of Sciences in Nantes.
They were maintained at 22°C on a 12:12-h light-dark cycle
and provided with rodent chow and tap water ad libitum.
This study was conducted under an authorization (no.
A44601) for the use of laboratory animals accorded by the
French Department of Agriculture.
Synthesis of riboprobes. Plasmids C4E3, NC2, and P20,
containing inserts of rat brain NCX1, NCX2, and NCX3
cDNA, respectively, were a generous gift from Ken Philipson
(University of California, Los Angeles, CA). The C4E3 plasmid contained a 520-bp fragment of NCX1 cDNA (630–1150
bp of the coding region) inserted into the EcoR I site of
pBluescript SK⫹. The NC2 plasmid contained an insert
corresponding to the coding region of NCX2 from 1916 to
2422 bp. The P20 plasmid contained an insert in the EcoR I
site corresponding to 2017 bp of the initial coding region of
NCX3. To eliminate portions of the pBluescript SK⫹ flanking
the T7 promoter, the plasmids were treated with EcoR V and
Kpn I, blunt-ended, and recircularized. The modified C4E3
was digested at the vector Sma I site, the NC2 at the BamH
I site, and the P20 with Cla I at the 1405-bp position of
NCX3. The linearized plasmids were electrophoresed in an
agarose minigel, extracted with a JETsorb gel extraction kit
(Genomed), and purified by phenol-chloroform extraction.
Digoxigenin-UTP-labeled antisense RNA probes were transcribed from the templates with T7 RNA polymerase (Boehringer Mannheim). The templates were removed by DNase
digestion. The antisense digoxigenin-labeled glyceraldehyde3-phosphate dehydrogenase (GAPDH) and 28S RNA probes
were synthesized with SP6 RNA polymerase from pTRI
GAPDH rat and pTRI RNA 28S templates (Ambion, Austin,
TX).
Northern blotting. RNA was isolated by the guanidiniumphenol-chloroform solvent extraction method (RNA Plus, Bioprobe Systems, Montreuil-sous-Bois, France), dissolved in
diethyl pyrocarbonate (DEPC)-treated water, and then diluted with ethanol and stored at ⫺80°C before use. The
amount and quality of the total RNA in samples were determined by measuring absorbance at 260 nm and 280 nm and
checked by ethidium bromide staining of 28S and 18S RNA in
minigels.
Twenty micrograms of total RNA were applied per lane of
formaldehyde-denatured 1% agarose gels. The electrode
buffer was supplemented with 2.2% formaldehyde. After
overnight electrophoresis, the gels were soaked for 20 min in
50 mM NaOH, washed with DEPC-treated water, and impregnated for 45 min with 10⫻ SSC (1⫻ SSC: 0.15 M
NaCl-15 mM sodium citrate, pH 7.0). The upward capillary
transfer to nylon membranes (Boehringer Mannheim) was
performed in 10⫻ SSC for 3–5 h; during this time, paper
towels were frequently changed. The membranes were baked
at 120°C for 30 min and prehybridized for 3 h at 68°C or 65°C
in the solution containing 50% formamide, 5⫻ SSC, 0.2%
SDS, 0.1% N-lauroylsarcosine, and 4% of the blocking reagent (Boehringer Mannheim). Hybridization with digoxigenin-labeled riboprobes was performed overnight under the
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coexistence of fast and slow fibers with a predominant
glycolytic or oxidative metabolism, it might be expected
that some cells differ in their dependence on extracellular Ca2⫹ and thus in their Na⫹/Ca2⫹ exchange activity.
All exchanger isoforms have a similar organization
(38), consisting of 11 transmembrane segments and a
large intracellular loop providing secondary regulation
of the exchange activity by Ca2⫹. Elimination of the
intracellular loop by ␣-chymotrypsin treatment of
membrane vesicles obtained from transfected BHK
cells considerably increases the activity of the three
exchanger isoforms (25). NCX1 contains a high-affinity
Ca2⫹-binding domain composed of about 150 amino
acid residues (22, 29). Two Ca2⫹-binding sites have
been identified within this segment, each characterized by the presence of three successive aspartate residues (21, 22), an unusual arrangement for members of
the large family of Ca2⫹-binding proteins (27). Although the exchanger isoforms share no more than
70% identity, the two acidic sequences forming the
high-affinity Ca2⫹-binding domain of the NCX1 (22,
29) are conserved in NCX2 and NCX3.
The study of NCX1 and NCX3 isoforms in skeletal
muscle is of particular interest because alternative
processing of their transcripts gives rise to multiple
variants and because the expression of some of the
splicing variants seems to be developmentally regulated (38). The variable region of the NCX1 gene (located in the carboxyl end of the intracellular loop)
contains two mutually exclusive exons, A and B, and
four cassette exons, C to F (16). In shorter NCX2 and
NCX3 isoforms, this region is organized in sequences
AC and ABC, respectively. Analysis of the transcripts
in neonatal and adult rat skeletal muscles by reverse
transcriptase-polymerase chain reaction (RT-PCR) has
revealed six splicing isoforms of NCX1 and two of
NCX3 (38). To date, no attempt has been made to
quantify the expression of splicing exchanger isoforms
or to estimate their relative contribution to total Na⫹/
Ca2⫹ exchange activity in skeletal muscle. Some of the
numerous exchanger variants can be expressed in different types of muscle, and their expression may vary
during development.
The developmental approach has been quite fruitful
in elucidating the functional significance of Na⫹/Ca2⫹
exchange in myocardial excitation-contraction coupling (4, 6, 41). It has been determined that the NCX1
isoform is expressed at much higher amounts in newborn than in adult cardiomyocytes (4, 41). A downregulation of NCX1 expression during postnatal stages,
correlated with a decrease in Na⫹/Ca2⫹ exchange activity in isolated sarcolemmal vesicles, occurs after a
progressive increase in SR Ca2⫹ pump activity (41).
These data indicate that Na⫹/Ca2⫹ exchange plays a
greater role in excitation-contraction coupling in immature than in adult heart (6).
The present study shows that the expression of the
Na⫹/Ca2⫹ exchanger in rat skeletal muscles is also
developmentally regulated. The levels of NCX1 mRNA
and protein increased up to 3 days postnatally and
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DHP receptors. The DHP receptors were visualized using
fluorescent-labeled DHP [(⫺)-DM-BODIPY-DHP, Molecular
Probes]. Longitudinal sections were postfixed for 20 min in
4% paraformaldehyde. Nonspecific sites were blocked for 30
min in PBS supplemented with 2% fetal calf serum. The DHP
receptors were revealed after a 30-min incubation in fluorescein-conjugated 0.1 ␮M DHP in PBS containing 2% calf
serum.
RESULTS
Expression of NCX1 mRNA in developing skeletal
muscles. RNA was isolated from the hind-leg muscles
of WKY rats. Whole hindlimb muscles were removed
from 1- to 16-day-old animals. Slow-twitch (soleus) and
fast-twitch (tibialis and EDL) muscles were excised
from 26-day-old and 7-wk-old rats. Hybridization of the
total RNA isolated from skeletal muscle with an NCX1
digoxigenin-riboprobe revealed a 7.5-kb band (Fig. 1)
corresponding to a major transcript of the cardiac exchanger (19). Moreover, a smaller transcript was found
in heart and skeletal muscles. This transcript, which
was not present in purified mRNA preparations, seems
to correspond to a recently described 1.8-kb circularized transcript lacking poly(A) (23).
The expression of the 7.5-kb NCX1 transcript in
developing hindlimb muscles progressively decreased
to quite reduced levels in 7-wk-old fast-twitch (EDL,
tibialis) and slow-twitch (soleus) muscles. By that time,
the level of NCX1 mRNA in slow-twitch muscle had
become lower than that in fast-twitch muscles.
Immunodetection of NCX1 isoforms. In our previous
studies on rat tissue homogenates, it was determined
that R3F1 antibody raised against dog heart NCX1
reacted in Western blots with two major exchanger
Fig. 1. Relative Na⫹/Ca2⫹ exchanger isoform NCX1 mRNA contents
in rat skeletal muscles. The positions of RNA-digoxigenin size standards (Stratagene) in kb (left) and of 28S and 18S RNA (right), as
revealed by ethidium bromide staining, are shown. To normalize
NCX1 levels, the blot was rehybridized with 28S RNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probes. EDL, extensor
digitorum longus; d, day.
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same conditions. The membranes were washed at room temperature with 2⫻ SSC-0.1% SDS and twice for 15 min at
69°C with 0.1⫻ SSC-0.1% SDS. Treatment of Northern blots
with alkaline peroxidase-conjugated anti-digoxigenin antibody and disodium 3-(4-metoxyspiro{1,2-dioxetane-3,2⬘-(5⬘chloro)tricyclo[3.3.113,7]decan}-4-yl) phenyl phosphate, a
chemiluminescent substrate, was performed according to the
manufacturer’s recommendations (Boehringer Mannheim).
The wet nitrocellulose membranes were sealed in plastic
bags and stored at 4°C or ⫺20°C before rehybridization with
28S RNA and GAPDH riboprobes.
Tissue homogenates and Western blots. One hundred milligrams of frozen rat tissues were homogenized in an UltraThurrax in 1 ml of medium containing 20 mM HEPES (pH
7.0), 1 mM sodium azide, 1 mM phenylmethylsulfonyl fluoride, 1 ␮g of aprotinin, and 1 mM dithiothreitol. The crude
homogenates or supernatants obtained after a 20-min centrifugation at 700 g were frozen in liquid nitrogen and stored
at ⫺80°C. Protein concentration was determined by a protein
quantification kit (Bio-Rad, Yvry Sur Seine, France) using
bovine serum albumin as standard.
Laemmli SDS-PAGE and electrotransfer of the proteins to
nitrocellulose membranes (Bio-Rad) were done as previously
described (21). The membranes were blocked for 1 h with a
solution of 150 mM NaCl, 10 mM Tris 䡠 HCl (pH 7.6), 5% skim
milk powder, and 0.1% Tween 20, preincubated for 1 h with
R3F1 monoclonal antibody against dog heart NCX1 (36) or
with a polyclonal antibody against rat brain NCX2 and
NCX3, and treated with peroxidase-linked anti-mouse
F(ab⬘)2 fragments (Amersham, Les Ulis, France) or goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratories,
West Grove, PA), respectively. The blots were developed with
an enhanced chemiluminescence detection kit (Amersham)
and exposed to X-Omat film (Kodak, Rochester, NY).
Histochemical and immunocytochemical analysis. Soleus
and extensor digitorum longus (EDL) muscles were excised,
stretched approximately to rest length, and quickly frozen in
precooled isopentane. Transverse serial cryostat sections (6
␮m) were cut from the muscle midregion, collected on gelatin-coated slides, and air-dried for 1 h at room temperature.
Alternate slides were used to examine activities of myofibrillar ATPase, NADH-tetrazolium reductase [oxidative enzyme
activity, NADH-tetrazolium reductase (TR)], immunostaining of NCX1, or staining of the dihydropyridine (DHP) receptor.
Tissue sections were fixed for 20 min in acetone at 4°C,
kept in blocking solution (2% fetal calf serum and 5% goat
serum) for 1 h, and then incubated overnight at 4°C with
R3F1 monoclonal antibody against NCX1 or with polyclonal
antibody against NCX3 diluted 1:50 or 1:1,000, respectively,
in phosphate-buffered saline (PBS). The secondary antibodies (fluorescein-labeled goat anti-mouse IgG1 for NCX1 and
goat anti-rabbit IgG for NCX3 diluted 1:100 in PBS) were
applied on sections for 1 h at room temperature. Control
experiments were performed using serial sections treated
only with the secondary antibody diluted 1:100 in PBS for 1 h
at room temperature. After several washes in PBS, the sections were mounted in 70% glycerol in PBS. Fluorescent
images were observed with a Leica Aristoplan microscope
and photographed using Kodak film (400 ASA).
Myofibrillar ATPase assays were performed according to a
previously described method (5). Briefly, sections were preincubated at room temperature either in an acid buffer (pH
4.2) for 10 min or in an alkaline buffer (pH 10.4) for 20 min.
The sections were then incubated in a buffer (pH 9.4) containing 4 mM ATP at 37°C for 20 min. NADH-TR was
revealed using NADH and nitro blue tetrazolium (9).
NA⫹/CA2⫹ EXCHANGER DURING MUSCLE DEVELOPMENT
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bands of 120 and 100 kDa (21). The first NCX1 variant
was predominant in heart and the second in lung and
some other tissues, whereas both were present in
brain. Figure 2 shows that these two exchanger variants of low and high electrophoretic mobilities (NCX1L
and NCX1H, respectively) are expressed in rat skeletal
muscles. Brief preincubation of brain, heart, and skeletal muscle homogenates in electrophoresis sample
buffer supplemented with EGTA gave a single band
with an intermediate molecular mass of nearly 110
kDa.
Important changes in NCX1L and NCX1H expression occurred in differentiating skeletal muscle. The
NCX1H variant, which is predominant in newborn
muscle, practically disappeared by the 7th wk in the
tibialis and EDL but was still present in slow-twitch
muscle (soleus), although at a much lower concentration than that present after birth. Figure 2 shows a
different expression for NCX1L protein, which was also
developmentally regulated. By the 7th wk, NCX1L was
still easily detectable in fast-twitch muscle, but present
only in trace amounts in slow-twitch muscle.
It is noteworthy that the decrease in NCX1 protein
expression in developing rat skeletal muscle (Fig. 2)
was closely related to the decrease in message abundance (Fig. 1).
Intracellular localization of NCX1. Immunofluorescence studies were performed on 3-day-old and adult
rat muscle fibers to localize NCX1 in skeletal muscle.
In 3-day-old muscle, which presumably lacked a welldeveloped T-tubular system, NCX1 was highly expressed on surface membrane compared with intracellular staining (Fig. 3). Three major types of muscle
fibers (type 1, slow oxidative; type 2A, fast oxidativeglycolytic; and type 2B, fast glycolytic) were revealed
histochemically in mature muscle by combined stain-
ing for myosin ATPase (33) and oxidative and glycolytic
enzyme markers (34). Figure 4 shows that adult rat
soleus is predominantly composed of type 1 fibers, with
few type 2A (a and b), whereas EDL represents a
mixture of 2A and 2B fibers (d and e). In both soleus
and EDL muscles, NCX1 was expressed predominantly
in 2A fibers, to a lesser extent in type 1, and very
weakly in 2B (Fig. 4, c and f). Clusters of NCX1 were
detected in some sarcolemma regions of 2A fibers (Fig.
4, c and f; Fig. 5a). The exchanger isoform was also
distributed more or less homogeneously in the form of
small spots within myofibers (Fig. 5a). Observations of
longitudinal sections at high magnification (Fig. 5b)
showed a cross-striated pattern of NCX1 immunostaining, possibly corresponding to the localization of T
tubules. In fact, a similar pattern was obtained when
serial sections were treated with fluorescein-conju-
Fig. 3. Transverse muscle sections from a 3-day-old rat treated with
R3F1 MAb. All myofibers exhibit fluorescent labeling of their sarcolemma (a and b). Vessel walls are highly labeled with R3F1 MAb (a,
arrowheads). Magnification: a, ⫻125; b, ⫻400.
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Fig. 2. Western blots of homogenates isolated from skeletal (sk) muscle, brain, and heart of rats of different ages.
Twenty (skeletal muscle) and ten (brain, heart) micrograms of the total protein of each sample were placed in a
sample buffer supplemented with 9% ␤-mercaptoethanol and 2 mM CaCl2 or 2 mM EGTA. The proteins were then
separated in 9% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad) for 45 min at 100
V. The position of prestained markers (Bio-Rad) is indicated. NCX1 expression was revealed by R3F1 monoclonal
antibody (MAb) raised against canine cardiac exchanger (see EXPERIMENTAL PROCEDURES). Two forms of NCX1 were
detected in gels after pretreatment of samples with Ca2⫹: NCX1H and NCX1L, with higher (H) and lower (L)
electrophoretic mobility, respectively (as indicated by dashes). Note that the bands move in the opposite direction
in the presence of EGTA and that their apparent molecular masses become almost identical. The mobility of
exchanger proteolytic products in gel was also modified after EGTA pretreatment.
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gated DHP (Fig. 5c), a marker of the triads formed by
membranes of junctional SR and T tubules.
Expression of NCX2 and NCX3 exchangers. As indicated by Northern and Western blot analyses, NCX1
expression in skeletal muscle was downregulated during postnatal development. Because the level was significantly decreased by the seventh postnatal week, it
is likely that Na⫹/Ca2⫹ exchange plays a minor role in
adult rat muscle. However, two other exchanger isoforms (NCX2 and NCX3) may contribute to the activity
of muscle cells.
As shown in Fig. 6, the NCX2 transcript of 5 kb was
present in the brain of rats of different ages. However,
no signal was detected in developing muscle under
high (68°C), low (60°C), or intermediate (65°C, Fig. 6)
hybridization and washing stringencies or after a
longer exposure time. A small 1.4-kb transcript reported previously (32) was also undetectable. These
results were supported by Western blot analysis of
NCX2 expression in the adult rat. Similar to the NCX1
protein (Fig. 2), NCX2 in brain changed its electrophoretic mobility after samples were pretreated in the
presence of EGTA (data not shown). Very faint signals
were detected on the blot containing heart and muscle
samples (Fig. 7), which seem to result from nonspecific
Fig. 6. Northern blot of rat brain and skeletal muscle mRNA hybridized successively with NCX2 and GAPDH probes. Prehybridization
and hybridization were performed at 65°C. The positions of 28S RNA
and 18S RNA (ethidium bromide staining) (left) and of digoxigeninlabeled RNA markers (in kb; right) are also indicated.
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Fig. 4. Serial transverse sections of adult rat soleus (a–c) and EDL
(e–f) muscles (⫻115). Sections were treated at pH 10.4 (a and d) to
reveal myofibrillar ATPase activity. b and e: NADH-TR activity. c
and f: Immunocytochemical reactivity with R3F1 MAb. NCX1 was
detected in type 1 and 2A fibers in both soleus and EDL muscles,
although the labeling of type 2A was more marked. Type 2B fibers in
EDL were weakly stained with R3F1 MAb.
Fig. 5. Subcellular localization of NCX1 in adult EDL type 2A
muscle fibers. Transverse (a) and longitudinal sections (b) were
treated with R3F1 MAb. Immunofluorescence staining indicates the
presence of NCX1 within fibers (a) and in certain areas of sarcolemmal membrane (a, arrowheads; ⫻400). High magnification of a
longitudinal section treated with R3F1 reveals a cross-striated pattern (b) similar to that visualized after staining with a fluorescentlabeled dihydropyridine (c).
NA⫹/CA2⫹ EXCHANGER DURING MUSCLE DEVELOPMENT
Fig. 7. Western blots of NCX2 from adult rat tissues. Seventy-five
micrograms of 700-g supernatants were incubated in loading buffer
in the presence of 1 mM CaCl2 or 2 mM EGTA, loaded into 13.5-mmwidth wells, and electrophoresed in 8% polyacrylamide gels. Note
that the position of the polypeptides of muscle and heart samples
does not change after EGTA treatment.
Northern blots in adult rat aorta (Fig. 8), heart, spleen,
lung, and kidney (data not shown).
Polyclonal anti-NCX3 antibody revealed similar developmental changes in the third exchanger isoform
(Fig. 9). Similar to the other two isoforms, the mobility
of NCX3 and of shorter polypeptides (presumably corresponding to proteolytic products of the protein) was
modified when electrophoresis sample buffer was supplemented with EGTA.
In 3-day-old muscles, no immunocytochemical reactivity was detected with anti-NCX3 antibody (data not
shown). In oxidative fibers of adult soleus muscle (83%
of type 1 and 17% of type 2A) (2, 12), NCX3 was
preferentially localized in the sarcolemma (Fig. 10,
a–c). In adult EDL muscle, the fast glycolytic fiber
population (type 2B) reacted much more strongly with
anti-NCX3 than did 2A fibers or very rare type 1 fibers
(Fig. 10, d–f). The staining of 2B fibers was especially
intense in myoplasmic space.
DISCUSSION
Immunologic and Northern blot techniques were
used to study Na⫹/Ca2⫹ exchanger expression in developing slow- and fast-twitch skeletal muscles. The
expression of two exchanger isoforms (NCX1 and
NCX3) in differentiating fibers was regulated in a
reciprocal manner. NCX1 was observed at a much
greater amount in newborns than in adults, which is
consistent with developmental changes occurring in
rabbit and rat hearts (4, 41). Quednau et al. (38), using
a semiquantitative RT-PCR technique, failed to detect
any differences in NCX1 expression between newborn
and adult hearts but found expression in newborn
skeletal muscle to be lower than that in adult. These
authors noted that expression at the protein level
needs to be investigated to exclude “illegitimate transcription” of genes easily detected by PCR.
Our results show that adult slow-twitch muscle and
fast-twitch muscle express distinct variants of NCX1
Fig. 8. NCX3 mRNA expression in developing rat brain and skeletal muscle
and its levels in adult rat muscle.
Twenty micrograms of total RNA were
applied per lane. A portion of the second blot was rehybridized with 28S
digoxigenin-labeled probe.
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binding of the polyclonal antibody, since the corresponding polypeptides were not sensitive to EGTA pretreatment. Together, these results for Western and
Northern blot analyses suggest the absence of musclespecific NCX2 expression.
Figure 8 shows Northern blots of NCX3 mRNA from
developing muscles of WKY rats and different muscles
of an adult WKY rat. This exchanger isoform seems to
be of particular interest because its expression in the
rat was found to be restricted to brain and skeletal
muscle, with two spliced variants identified in muscle
tissues (38). Comparison of NCX3 expression in muscle
tissues and rat brain showed that its transcript (6 kb,
just above the band of 28S RNA) was present at a
similar level in brain from all age groups and tended to
decrease in the adult (6-mo-old) rat. Conversely, NCX3,
although practically absent in skeletal muscle of newborn rats, increased dramatically by the second week of
postnatal development. No difference was found between NCX3 expression in slow-twitch (soleus), fasttwitch (tibialis, EDL, gastrocnemius), and mixed (diaphragm) muscles. No NCX3 transcript was detected by
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NA⫹/CA2⫹ EXCHANGER DURING MUSCLE DEVELOPMENT
Fig. 9. NCX3 protein expression in developing rat skeletal muscle.
The blot was treated with polyclonal anti-NCX3 antibody. Other
conditions were as indicated in Fig. 7. diaph, Diaphragm.
Fig. 10. Serial sections of adult rat soleus (a–c) and EDL (d–f)
muscles (⫻115). Sections were treated at pH 10.4 (a and d) to reveal
myofibrillar ATPase activity. b and e: NADH-TR activity. c and f:
Immunocytochemical reactivity with anti-NCX3 antibody. In type 1
and 2A fibers from both muscles, NCX3 was preferentially detected
at the sarcolemma level. NCX3 was highly expressed in myoplasmic
space of type 2B fibers.
Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on May 5, 2017
polypeptide (NCX1H and NCX1L, respectively), which
differ in their electrophoretic mobility. This difference
was detected only after pretreatment of electrophoresis
samples in the presence of Ca2⫹, which may indicate
that regulatory Ca2⫹ binding sites in the hydrophilic
loop of the exchanger were saturated (22). Because the
process of Ca2⫹ interaction with the high-affinity Ca2⫹
binding domain of NCX1 is highly cooperative (21),
conformational changes may occur in the NCX1
polypeptide on Ca2⫹ binding. These conformational
changes seemed to persist even in the presence of SDS,
as in the case of other high-affinity Ca2⫹-binding proteins (28). It is noteworthy that NCX1H, which is
highly expressed at birth when rat limb muscles are
known to be slowly contracting (for review, see Ref. 7),
shows markedly weaker expression later on (mainly in
fast-twitch muscle). NCX1H could be specific to slowly
contracting muscle fibers.
This study did not allow us to develop conclusions as
to the exact origin of NCX1H and NCX1L. A recent
report (23) showed differences in the electrophoretic
mobility of NCX1 polypeptides, corresponding to the
splicing of isoforms with exon combinations BD (apparent molecular mass of 110 kDa) and ACDEF (cardiac
variant, 120 kDa). The fact that amplification of cDNA
from adult rat fast-twitch skeletal muscle showed predominant expression of ACDEF (38) suggests that the
NCX1L observed in 7-wk-old rats may have corresponded to this cardiac variant. Four splicing isoforms
(BDEF, BDF, BCD, BD) have been identified in neonatal muscle (38). Thus additional information is needed
to determine the exact composition of the broad
NCX1H band on the blot (Fig. 2).
The possible involvement of the Na⫹/Ca2⫹ exchange
in skeletal muscle excitation-contraction coupling was
supported by our finding that NCX1 is not evenly
distributed but clustered in sarcolemmal membrane.
These clusters of NCX1 could modify Ca2⫹ levels in
some subsarcolemmal regions, leading to rapid extrusion of the ions from muscular cells and/or affecting
Ca2⫹ channels of sarcolemma and peripheral junctional SR. It is still unclear whether this NCX1 arrangement corresponds to clusters of DHP receptors
and/or ryanodine receptors of the SR Ca2⫹ release
channel, as described for avian muscle cells (37), vas-
cular smooth muscle cells (14), and amphibian smooth
muscle cells (31).
Electrophysiological data suggest that Na⫹/Ca2⫹ exchange may be of greater functional importance in
slow-twitch skeletal muscles (13) and in the diaphragm
(45) than in fast-twitch muscles possessing a welldeveloped SR network. Surprisingly, we detected
higher levels of NCX1 expression in rat fast-twitch
(EDL, tibialis) than in slow-twitch (soleus) muscle.
NCX1 protein was predominantly expressed in oxidative fibers (types 1 and 2A) in both soleus and EDL
muscles, and the intensity of immunostaining was
greater in type 2A than in type 1. Thus it is likely that
the relatively higher NCX1 protein and mRNA levels
found in rat fast-twitch muscle were due to the predominance of 2A fibers. In the adult rat, EDL is composed of 40% fast oxidative-glycolytic (type 2A) and
60% fast glycolytic fibers (type 2B), whereas soleus
contains ⬃17% type 2A and 83% slow oxidative fibers
(type 1) (2, 12). If we consider the expression of NCX1
at the level of each fiber type, our results may relate to
functional differences between muscle fiber phenotypes. The very low amount of NCX1 in type 2B fibers,
which are characterized by a very well-developed SR
network and a high capacity for SR Ca2⫹ uptake (15,
NA⫹/CA2⫹ EXCHANGER DURING MUSCLE DEVELOPMENT
specific roles of NCX1 and NCX3 isoforms. We have
shown that the expression of NCX1 and NCX3 differs
greatly in 2A and 2B fibers. Thus it would seem worthwhile investigating the contractile responses of isolated 2A and 2B fibers to variations in extracellular
Na⫹ concentration. Alternatively, the effects of NCX1
and NCX3 anti-sense oligonucleotides on contractile
properties of muscle fibers and/or cytoplasmic free
Ca2⫹ concentration could be checked under conditions
favoring a normal or reverse mode of Na⫹/Ca2⫹ exchange. Further studies on isolated muscle fibers as
well as on cultured muscle cells are required to address
these questions.
We are grateful to Dr. K. D. Philipson (UCLA) for generously
supplying R3F1, anti-NCX2, and NCX3 antibodies and plasmids and
to Yvonnick Chéraud and Felix Crossin for technical assistance.
This work was supported by the University of Nantes, le Centre
National de la Recherche Scientifique, and l’Association Française
contre les Myopathies.
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