Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital, Boston, Massachusetts
Submitted 20 September 2004 ; accepted in final form 11 November 2004
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ABSTRACT |
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ryanodine receptor; calcium release; ryanodine binding; muscle fibers
The use of animal models null for all RyR isoforms has been of particular help in uncovering the specific role that each of the RyR isoforms plays in normal muscle physiology. Studies of cells from RyR1- or RyR3-knockout mice have shown that unlike the absence of RyR1, the lack of RyR3 does not result in alteration of electrically evoked Ca2+ release or the contractile function of adult mammalian skeletal muscle fibers (3, 34). In addition, in mouse diaphragm, one of the mammalian muscles that has the highest expression levels of RyR3, there have been no alterations in excitation-contraction (EC) coupling or in the ability of this muscle to contract detected in mature fibers from null mice (1, 5, 9). Moreover, expression of recombinant RyR3 in dyspedic 1B5 myotubes, which do not express any of the RyR isoforms, failed to restore either electrically stimulated or K+-induced Ca2+ release (13, 26), suggesting that RyR3 has virtually no role in initiating or maintaining skeletal EC coupling.
Significant functional differences between RyR3 and RyR1 are observed in their sensitivity to caffeine and 4-CmC, two known direct RyR agonists. Studies in dyspedic 1B5 myotubes have shown that cells expressing RyR3 have a lower threshold and EC50 for caffeine than do cells expressing RyR1 (13, 26), a property that was also observed when these two isoforms were expressed in human embryonic kidney (HEK)-293 cells (28). To the contrary, myotubes expressing RyR1 have a significantly higher sensitivity to 4-CmC than do those expressing RyR3, which has almost no 4-CmC response at all, even at millimolar concentrations (12, 13). These differences suggest that in situ both RyR1 and RyR3 have a characteristic and differential sensitivity to direct agonists.
Despite very low levels of RyR3 in adult mammalian muscle relative to RyR1, a transient increase in expression of RyR3 during postnatal muscle development has been reported (3, 35). Although such variation in expression levels of RyR3 is likely to cause a significant effect on Ca2+ signaling in skeletal muscle, no systematic studies have been performed to correlate these two events. Studies in neonatal mouse skeletal myotubes null for RyR3 have shown that the presence of RyR3 in wild-type (wt) myotubes amplified the Ca2+-induced Ca2+ release (CICR) signal induced by RyR1 (40) and improved the electrical and caffeine-evoked muscle contracture (3, 7). In addition, expression of RyR3 has been associated with augmented spontaneous Ca2+ activity in muscle fibers and cultured myotubes, as well as with increased frequency and size of Ca2+ sparks (6, 7, 37, 38). Although these studies did not define the role of RyR3 in muscle cells, they clearly suggested that the expression of RyR3 in mammalian skeletal muscle, although small compared with RyR1, could play a significant role in Ca2+ homeostasis. The gaps in information regarding the function of RyR3 stress the need for a comprehensive understanding of the functional properties of RyR3 and the role of RyR3 in Ca2+ homeostasis in skeletal muscle.
In the present study, we sought to further characterize the functional differences between RyR1 and RyR3 in myotubes and muscle fibers. Using recombinant RyRs expressed in dyspedic 1B5 myotubes as well as in neonatal muscle fibers, we assessed the effects of variation in expression levels of each isoform on the physiology of cultured myotubes and muscle fibers. Our results show that expression of either isoform had a profound effect on myoplasmic resting free Ca2+ and caffeine sensitivity of the myotubes. Expression of RyR3 always resulted in myotubes with resting free Ca2+ concentration ([Ca2+]r) that was significantly higher, as well as EC50 for caffeine that was significantly lower, than myotubes expressing RyR1. Similarly, in both postnatal and adult skeletal muscle fibers that expressed RyR3, myoplasmic [Ca2+] increased proportionally to the levels of RyR3. These results show that both caffeine sensitivity and increased [Ca2+]r induced by the expression of RyR1 or RyR3 are not the result of variations in expression level of the channels but arise from intrinsically distinct properties that are characteristic of each isoform.
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MATERIALS AND METHODS |
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Membrane preparation. Crude membranes were prepared from six to eight 100-mm plates of 1B5 myotubes 36 h after transduction with virion particles. Each plate was infected with either 1.0, 2.0, 5.0, or 20.0 x 106 virion particles, which corresponded to MOI equivalent to those used in the 96-well plate format for imaging. Myotubes were harvested as described previously (26).
Neonatal tibialis anterior (TA) muscles were dissected from mice at postnatal days 5, 10, and 15, while adult TA and soleus muscles were harvested at postnatal day 45. All collected muscles were quick-frozen in liquid N2. Microsomal vesicles were prepared by homogenization in a Polytron cell disrupter (Brinkmann Instruments, Westbury, NY) in buffer consisting of 20 mM HEPES, pH 7.0, supplemented with 250 mM sucrose and protease inhibitor cocktail (Complete; Roche, Indianapolis, IN). Whole homogenates from cultured cells and muscles were centrifuged at 1,500 g, and the supernatants were collected and recentrifuged at 100,000 g. The collected membranes were resuspended in 250 mM sucrose and 20 mM HEPES, pH 7.4; frozen in liquid N2; and stored at 80°C. Junctional sarcoplasmic reticulum (JSR) vesicles used as controls were obtained from rabbit skeletal muscle according to the method described by Hidalgo et al. (15).
Gel electrophoresis and immunoblotting. SDS-polyacrylamide gel electrophoresis (18) was performed on proteins from crude homogenates as described above. Immunoblots were incubated with monoclonal antibody (MAb) 34C (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), which recognizes both RyR1 and RyR3 isoforms, or a polyclonal anti-RyR3 antibody that specifically recognizes RyR3 (gift from Dr. T. Murayama). Identification of FK506-binding protein (FKBP-12) was achieved using polyclonal antibody PA1-026A, while sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)1 and triadin were detected with MAb MA3-911 and MA3-927 (Affinity Bioreagents, Golden, CO), respectively. Calsequestrin was detected with a COOH-terminal polyclonal antibody that recognizes both skeletal and cardiac calsequestrin (gift from Dr. L. Jones). Membranes were then incubated with either anti-mouse or anti-rabbit secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, West Grove, PA). Immunoreactive proteins were developed with SuperSignal enhanced chemiluminescence substrate (Pierce Biotechnology, Rockford, IL).
[3H]ryanodine binding assay. Maximal high-affinity [3H]ryanodine binding to crude membrane extracts (0.1 mg/ml) was performed at equilibrium in the presence of 1 M KCl, 100 µM free Ca2+, 20 mM HEPES, pH 7.2, and 10 nM [3H]ryanodine (PerkinElmer Life Sciences, Boston, MA) in the presence of a cocktail of protease inhibitors (Complete). Caffeine dose-response measurements were performed at 0.5 M KCl, 20 mM HEPES, pH 7.4, and 10 nM [3H]ryanodine, with free [Ca2+] adjusted to 500 nM (2). Nonspecific binding was determined by incubating the vesicles with 5 µM unlabeled ryanodine. Separation of bound and free ligand was performed by rapid filtration through GF/B glass fiber filters (Whatman, Middlesex, UK) using a cell harvester (Brandel, Gaithersburg, MD). Filters were washed with ice-cold wash buffer containing 20 mM Tris·HCl, pH 7.4, and placed into vials with 5 ml of scintillation cocktail. The [3H]ryanodine remaining on the filters was quantified using liquid scintillation spectrometry.
Resting free Ca2+ measurements. [Ca2+]r measurements in cells were performed in myotubes differentiated in 96-well plates as described above. Determination of [Ca2+]r in muscle fibers were performed in situ to avoid muscle hypoxia during measurement. Mice were anesthetized with ketamine and xylazine (50 and 7 mg/kg, respectively). TA and soleus muscles were exposed by dissection from the surrounding connective tissue and then were immersed in Hanks' balanced salt solution (GIBCO, Carlsbad, CA) supplemented with either 2 or 10 mM CaCl2.
Double-barreled Ca2+-selective microelectrodes were prepared as described previously (19). Microelectrodes were assembled using ETH129 resin, which has greater sensitivity to low [Ca2+] compared with ETH1001 (20). Electrodes were calibrated individually, and only those electrodes that had a Nernstian response between pCa3 and pCa7 (28.5 mV/pCa U at 24°C) were used experimentally. To prevent artifacts due to changes in the sensitivity of the microelectrode, individual Ca2+-selective microelectrodes were used for only five or six determinations. If the postexperiment recalibration curve did not agree within 2.5 mV/decade of the initial calibration between pCa6 and pCa8, the data were discarded. Single-myotube impalements were performed using an inverted microscope (Axiovert 10) at x160 magnification. Because the plasma membrane resting potential was acquired simultaneously with the Ca2+ potential during the entire experiment, any cell damage due to the impalement could be detected quickly. Therefore, all recordings in which Vm was less negative than 55 were discarded (normal range in myotubes is 60 to 55 when measured with 3 M KCl microelectrodes). To rule out the possibility of any potential contribution of extracellular Ca2+ leakage to the measured intracellular [Ca2+]r, a series of control determinations were performed in muscle fibers with two different concentrations of extracellular Ca2+. The potentials from both the 3 M KCl barrel (Vm) and the Ca2+ barrel (VCae) were acquired with high-impedance amplifiers (WPI FD-223). Vm was subtracted electronically from the VCae to obtain a differential signal (VCa) that represented the myoplasmic [Ca2+]r. Vm and VCa potentials were filtered at 200 Hz (WPI-LPF-30) and were analyzed using AxoGraph version 4.8 software.
Data analysis. Ca2+ imaging data were analyzed as described previously (25). Briefly, to compare data from different experiments, fluorescent Ca2+ transients induced by any given caffeine concentration were normalized to the peak amplitude of the maximal response to caffeine from the same cell. Caffeine EC50 for each condition was calculated by fitting a logistic dose-response curve to individual dose responses. Statistically significant differences among the data were evaluated using one-way Kruskal-Wallis ANOVA (nonparametric) (GraphPad Software, San Diego, CA). Data are presented as means±SD or as means±SE.
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RESULTS |
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Immunopattern of expressed RyRs. To evaluate whether the increased levels of RyR expression had any effects on the subcellular organization of the expressed receptors, we analyzed the immunopattern of expression of myotubes infected with >10-fold differences in MOI. Myotubes were differentiated on coverslips and infected with either RyR1 or RyR3 at MOI 0.4 and 4.0, stained with MAb 34C, and analyzed with a x100 oil-immersion lens objective. As previously reported (27), three main immunopatterns of expression were readily identified in all groups of cells. These patterns included 1) a punctate pattern, in which the immunostaining appeared as defined regular foci of fluorescence, 2) a reticular pattern, defined as a network-like distribution with no recognizable foci of fluorescence, and 3) a punctate-reticular pattern, a combination of the two preceding patterns. Analysis of immunostained myotubes revealed that the three patterns of expression were always detected, regardless of the amount of virus used for infection (Table 1). At MOI 0.4, the punctate pattern was always predominant, accounting for 88.0% of the population of RyR1-expressing myotubes and 84.2% of RyR3-expressing cells. The punctate-reticular pattern, on the other hand, accounted for 10.4% of the RyR1-expressing myotubes and 12.7% of RyR3 population. The reticular pattern represented only 1.6% of RyR1- and 3.1% of RyR3-expressing cells. In myotubes infected at MOI 4.0, the punctate immunopattern of expression was still predominant for both isoforms, accounting for >60% of the cell population. However, at MOI 4.0, there was an increase in the percentage of punctate-reticular and reticular immunopatterns of expression in both RyR1- and RyR3-expressing cells. This was particularly apparent in cells overexpressing RyR3 (MOI 4.0), in which the punctate-reticular immunopattern was twofold and the reticular pattern was fourfold the percentage observed in myotubes infected at MOI 0.4. In addition, in myotubes overexpressing RyR3, the percentage was twofold that of the punctate-reticular and reticular immunopatterns in myotubes expressing RyR1 at similar MOI (Table 1).
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To analyze the expression levels of RyR1 and RyR3 at each stage of development, Western blot analysis was performed in studied muscles collected immediately after Ca2+ measurements. Crude membrane preparations from each muscle were obtained and analyzed with MAb 34C and anti-RyR3 antibodies to assess the total RyRs and RyR3, respectively. Figure 6A shows that although 34C antibody detected significant expression levels of total RyRs at all stages of TA muscle development, the adult muscles demonstrated only a small increase in total expression compared with the neonatal muscles. No significant variability in total RyR expression was evident between muscles from postnatal days 5 and 15 samples. However, there were significant variations in RyR3 expression among all tested stages. Figure 6A demonstrates that there was a trace of RyR3 expression in TA muscles at postnatal day 5, followed by stepwise increases in RyR3 expression at postnatal days 10 and 15 (3.5- and 4.5-fold increases, respectively, relative to 5-day-old muscle). There was no detectable RyR3 expression observed in adult muscles. Although this result is consistent with previous findings showing no detectable expression of RyR3 in microsome preparations of adult TA muscle (3), very low levels of RyR3 expression have been reported with the use of Western blot analysis and in situ hybridization in adult mouse and rat TA muscles (8).
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Effects of coexpression of RyR1 and RyR3 on [CA2+]R. Figure 7A summarizes the measurements of [Ca2+]r collected from several individual fibers of 610 muscles per group. As expected, a significant increase in [Ca2+]r was detected as the level of RyR3 expression increased in TA fibers. This increase was transient in nature because it declined as the animals reached maturity. Although no differences in average [Ca2+]r were evident between fibers from postnatal day 5 and adult muscles (107.3±4.8 and 111.8±3.0 nM, respectively; P > 0.05), a significant increase in average [Ca2+]r was observed in fibers from postnatal days 10 and 15 muscles (163.2±12.7 and 221.5±9.3 nM, respectively) compared with fibers from postnatal day 5 and adult muscles. On the other hand, measurement of [Ca2+]r in adult soleus muscle showed that [Ca2+]r in this muscle was significantly higher than that in adult TA fibers (166.4±8.6 vs. 111.8±3.0 nM, respectively; P < 0.005). Interestingly, just as in 1B5 myotubes, there was a wide variation in individual [Ca2+]r measurements, which we attributed to interfiber differences in the expression of RyR3. There was a large variation in individual [Ca2+]r measurements in TA fibers at postnatal days 10 and 15 and, although to a lesser extent, in adult soleus muscle, suggesting that there was a broad range of expression of RyR3 from fiber to fiber as suggested by Flucher et al. (14).
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DISCUSSION |
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In the present study, we have analyzed the effects of expression levels of RyR1 and RyR3 on Ca2+ response in 1B5 myotubes. It was established that over a broad range of RyR expression levels, there were very few differences in the overall phenotype of cultured myotubes expressing either isoform. The expression of either isoform had a substantial impact on caffeine sensitivity (both restored it) and [Ca2+]r (both increased it) compared with null myotubes. In addition, the caffeine sensitivity and [Ca2+]r measured in RyR3-expressing myotubes were significantly higher than the same parameters measured in RyR1-expressing cells. We also observed increases in [Ca2+]r in situ in postnatal days 10 and 15 TA muscle fibers and in adult soleus fibers. This difference appeared to be closely correlated with the expression levels of RyR3 observed at each stage.
Expression of RyR3 and its effect on myoplasmic [CA2+]R.
As viral MOI increased, we observed not only that a larger number of cells were transduced but also that a higher number of myotubes were infected by more than one viral particle, and thus more cells had the potential to overexpress their respective proteins. However, our Ca2+ microelectrode measurements demonstrated that RyR3-expressing myotubes had a cytosolic [Ca2+]r that, on average, was 2-fold that in myotubes expressing RyR1. Because total [3H]ryanodine binding was determined to be identical between RyR1- and RyR3-expressing cells at between MOI 0.2 and 1.0, it seems unlikely that the difference in [Ca2+]r resulted from the different levels of RyRs being expressed. Instead, this result suggests that the level of [Ca2+]r must strongly depend on the unique characteristics of each RyR isoform. On the basis of our measurements of resting free Ca2+ (Figs. 5 and 7), it is also clear that RyR3-expressing cells, in addition to having consistently higher [Ca2+]r than cells expressing only RyR1, both 1B5 myotubes and muscle fibers demonstrated wider dispersion of [Ca2+]r than did the cells expressing RyR1. It is our hypothesis that this phenomenon could be caused by the variable expression levels of RyR3 in individual myotubes or myofibers. This is consistent with immunohistochemical studies of developing murine muscle using anti-RyR3 antibodies, which have shown that there was an induction followed by a reduction of RyR3-expressing fibers during postnatal muscle development and that this induction followed by reduction does not take place gradually and simultaneously in all fibers, but instead occurs rapidly in some fibers and very slowly in others (14). Alternatively, the high variability of interfiber and myotube [Ca2+]r values may suggest that cells expressing RyR3 are variably hyperactive, constantly releasing puffs of Ca2+ into the cytosol. This hypothesis is consistent with studies of Ca2+ spark activity of RyR3-expressing cells that have indicated this to be the case. Interestingly, the high dispersion in individual [Ca2+]r measurements observed in our study in both myotubes and RyR3-expressing muscle fibers is in agreement with the report that embryonic myotubes that express RyR3 have considerably more variability in the size and kinetics of their Ca2+ sparks than do adult cells (7). This finding is also consistent with the fact that our [Ca2+]r data do not appear to reveal a defined population of fibers with either high or low [Ca2+]r as would be expected from muscles in which only some fibers express RyR3 and the others express only RyR1 (14). Significantly, at MOI 1.0, RyR3-expressing myotubes had [Ca2+]r values between 178 and 345 nM, a range similar to the 136363 nM concentration observed in 15-day-old TA fibers, which suggests that in terms of Ca2+ regulation, RyR3 in 1B5 myotubes demonstrated behavior similar to its behavior under physiological conditions. The importance of this result is that it 1) rules out the possibility that high [Ca2+]r detected in myotubes may be the result of toxic expression of RyR3 and 2) suggests that induction of high [Ca2+]r associated with RyR3 expression may be physiologically relevant.
Caffeine sensitivity of RyR1- and RyR3-expressing myotubes. It was reported previously that caffeine sensitivity of RyRs is highly Ca2+ dependent (30, 32, 41). However, it is unlikely that the higher [Ca2+]r induced by RyR3 was the only factor responsible for the differential caffeine sensitivity observed between RyR3- and RyR1-expressing cells in this study. This is supported by the [3H]ryanodine binding data in the present study, which demonstrated that there were significant differences in caffeine sensitivity between RyR1 and RyR3 even when the free [Ca2+] was fixed at a constant suboptimal level (500 nM). Nevertheless, under this condition, a significant difference in affinity to Ca2+ activation between both isoforms could also explain the divergence in caffeine sensitivity observed between RyR1 and RyR3, both in vivo and in vitro. Although it was previously suggested that RyR1 and RyR3 have a significant difference in Ca2+ sensitivity, these reports are somehow contradictory (see below). In this regard, [3H]ryanodine binding studies conducted under the same experimental conditions assayed in the present work indicated that RyR1 and RyR3 have very little difference in sensitivity for Ca2+ activation (Voss A., unpublished data). Thus it is highly unlikely that the difference in caffeine sensitivity in the present study is just the result of differential Ca2+ affinity between the two isoforms. Nevertheless, it is likely that the higher [Ca2+]r in RyR3-expressing cells was responsible for at least some of the increase in caffeine sensitivity observed in these cells.
The hypothesis that both an innate difference in caffeine sensitivity between the two isoforms and the increase in [Ca2+]r are responsible for the large difference in sensitivity observed in intact cells is supported by the fact that under the conditions used for binding, the difference in caffeine EC50 for RyR1 and RyR3 was only 2.5-fold (4.87±0.35 vs. 1.93±0.36 mM), which was significantly smaller than the 4.4-fold difference observed in intact cells infected at MOI 1.0 (3.54±0.34 vs. 0.79±0.19 mM). These results are in agreement with our previous findings (12, 13, 26) and those of others (6, 28, 29). On the basis of these results, it can be inferred that the differences in caffeine sensitivity observed in myotubes expressing RyR1 and RyR3 come from a true intrinsic difference between the two isoforms. Our data, however, are not in agreement with those of Murayama and Ogawa (23), who reported no difference in caffeine sensitivity between purified RyR1 and RyR3 isolated from bovine diaphragm. It is likely that this discrepancy arises from differences in the experimental conditions used in the two studies. The conditions the Murayama and Ogawa study were strongly activating, and thus it is possible that they any difference between the two isoforms may have been masked to further activation. Alternatively, the RyR3 isoform expressed in diaphragm may correspond to a different splice variant than the one expressed in uterus or in mouse skeletal muscle that were used in our present study. Recent studies in rabbit tissues have demonstrated that multiple splice variants of RyR3 are expressed in uterus and diaphragm and that some of these variants have significantly reduced caffeine sensitivity. Thus tissue-specific expression of RyR3 splice variants might offer an explanation for the heterogeneity of caffeine response of RyR3 in different tissues and cells (17).
Functional differences between RyR1 and RyR3. Several studies have revealed significant functional differences between RyR1 and RyR3, particularly in their Ca2+ and caffeine sensitivity. Several lines of evidence seem to support that RyR3 is less sensitive than RyR1 to Ca2+ inhibition (4, 16, 23, 33), although different experimental approaches have led various investigators to very different conclusions regarding its sensitivity to Ca2+ activation. Using single-channel analysis, Sonnleitner et al. (33) reported two populations of Ca2+ channels in SR vesicles from diaphragm muscle, one with properties similar to those of RyR1 and another, which they suggested was RyR3, that was less sensitive than RyR1 to Ca2+ activation. Consistent with this finding, [3H]ryanodine binding studies in purified receptors from diaphragm have consistently shown that RyR3 has a significantly lower sensitivity than RyR1 to Ca2+ activation (2123). However, this difference seems to be reversed when the receptors are fused into lipid bilayers in which Ca2+ EC50 for activation measured for RyR3 was found to be lower than that of RyR1 (21, 24). Studies of RyR3 from mouse parotid acini (10) and rabbit uterine RyR3 heterologously expressed in HEK-293 cells (4) have suggested that RyR3 has the same apparent sensitivity as RyR1 for Ca2+ activation. Whether these seemingly large differences in observed RyR3 Ca2+ sensitivity are due simply to different experimental conditions used in these studies or whether they reflect real intrinsic differences of various splice variants of RyR3 has yet to be resolved. A detailed analysis of the receptor splice variants at the single-channel level of RyR3 should be able to provide a conclusive answer.
In this regard, a detailed study of single-channel properties of the recombinant RyR3 used in the present work showed that although overall RyR3 shared several similarities with RyR1, at near-[Ca2+]r (200 nM) levels, RyR3 exhibited significantly longer mean open time (4) and an increase in subconductance behavior compared with RyR1 (13). These differences in gating kinetics between the two isoforms are consistent with the apparent higher Ca2+ leakage rates, which would lead to the higher [Ca2+]r that we observed in RyR3-expressing cells. This difference is also consistent with the fact that RyR3-expressing cells have a higher frequency and greater magnitude of Ca2+ sparks than do cells expressing only RyR1 (37, 38). Likewise, in studies of primary myotubes null for RyR3, a similar contribution of RyR3 to the magnitude (6, 7) and duration (31) of the Ca2+ sparks has been described, suggesting that important differences between the two isoforms exist with regard to Ca2+ release behavior. Our data support this hypothesis and suggest that this difference in spontaneous activity could be one of the major contributors to the difference in myoplasmic free Ca2+ that we observed between RyR1- and RyR3-expressing cells.
In summary, the results of this study demonstrate that the expression level of RyR3 in myotubes has a significant effect on myoplasmic free [Ca2+]r. These findings suggest that the coexpression of RyR3 in vivo at various levels might underlie a tightly regulated mechanism by which muscles fine-tune cytosolic free [Ca2+]. Such control would facilitate the diversity of cellular responses that muscle cells undergo during their early development.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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