Faculté des Sciences et des Techniques, Université de Nantes, Nantes Cedex 3, France
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ABSTRACT |
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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.
sodium/calcium exchanger isoforms; gene expression
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INTRODUCTION |
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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).
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 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 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.
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EXPERIMENTAL PROCEDURES |
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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 glyceraldehyde-3-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 guanidinium-phenol-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.
Tissue homogenates and Western blots.
One hundred milligrams of frozen rat tissues were homogenized in an
Ultra-Thurrax in 1 ml of medium containing 20 mM HEPES (pH 7.0), 1 mM
sodium azide, 1 mM phenylmethylsulfonyl fluoride, 1 µg 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.
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 µm) 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).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 µM DHP in PBS containing 2% calf serum.
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RESULTS |
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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).
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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 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.
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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 well-developed 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
oxidative-glycolytic; and type 2B, fast glycolytic) were revealed
histochemically in mature muscle by combined staining 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-conjugated
DHP (Fig. 5c), a marker of the triads formed by membranes of
junctional SR and T tubules.
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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 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 muscle-specific NCX2 expression.
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DISCUSSION |
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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 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), vascular 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 well-developed 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, 43), suggests that this isoform does not play a key role in intracellular Ca2+ regulation in fast glycolytic fibers. NCX1 protein was highly expressed in oxidative fibers, which displayed a less abundant sarcotubular system and a lower rate of shortening than type 2B fibers (Ref. 17; for a review, see Ref. 35).
No NCX2 expression was detected in developing or adult skeletal muscle at mRNA and protein levels, which makes the "NCX2 story" rather confusing. In fact, the initial report of Li et al. (24) concerning the presence of the 5-kb NCX2 transcript in skeletal muscle was not confirmed by the same group in a more recent publication (32). Instead, a 1.4-kb transcript was detected in a Northern blot of poly(A) RNA. More recently, Quednau et al. (38), using the far more sensitive RT-PCR technique, detected NCX2 transcripts in skeletal muscle as well as in a dozen other tissues. It is debatable whether the RT-PCR technique provides adequate quantitative information. In any event, the very high sensitivity of this technique should be considered because it may lead to amplification of transcripts present in nonmuscle cells. Because NCX2, which is expressed in brain, has a preferentially neuronal localization (38), some NCX2-specific RT-PCR products could originate at least partially from amplification of transcripts present in neurons (particularly motoneurons).
By the end of the second week after birth, there was a significant increase in the level of NCX3 transcripts, and a similar change occurred at the protein level. In serial sections of adult muscles, a definite correlation between fiber type and NCX3 content was demonstrated. Type 2B fibers displayed high staining intensity with antibody to NCX3, whereas type 2A and 1 fibers were weakly stained.
The results obtained in the present study point to important differences in the expression of NCX1 and NCX3. During postnatal development of skeletal muscle, NCX3 isoform expression increased markedly, whereas that of NCX1 decreased. This may indicate, in particular, that the Na+/Ca2+ exchange activity in adult muscle is governed predominantly by the NCX3 isoform and that NCX1 is more involved in regulating intracellular Ca2+ concentration in newborn skeletal muscle. This does not exclude involvement of the Na+/Ca2+ exchange mechanism in certain fibers of adult muscle. Indeed, NCX1 tends to form regular clusters in some membrane regions of type 2A fibers, and this may contribute to changes in the local Ca2+ level within the adjacent subsarcolemmal space.
However, additional experiments are required before conclusions can be reached about the specific roles of NCX1 and NCX3 in skeletal muscle. Several points need to be considered. One concerns the quantification of total Na+/Ca2+ exchange activity in different types of muscle. Unfortunately, comparative analysis of isoform expression at the protein level cannot be performed with currently available antibodies, especially because one of them (anti-NCX3) is polyclonal. Concerning the functional aspects of Na+/Ca2+ exchanger in muscle, our findings may help in understanding the 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* B. Fraysse and T. Rouaud contributed equally to this work.
This work was supported by the University of Nantes, le Centre National de la Recherche Scientifique, and l'Association Française contre les Myopathies.
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: dmitri.levitsky{at}svt.univ-nantes.fr).
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.
Received 6 July 1999; accepted in final form 17 July 2000.
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