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Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

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Related in: MedlinePlus

Depolarization-induced Ca2+ release.Intracellular Ca2+ ([Ca2+]i) of 1B5 myotubes transduced with either α-RyR or β-RyR virions was imaged using fluo-4 as described in Materials and Methods. Cells were exposed for 30 sec to varied high [K+]o solutions (with the constant [K+]×[Cl−] product) to trigger DICR, and finally to 20 mM caffeine to confirm the functional expression of RyR. Ca2+ in the bath solution was omitted prior to and during stimuli to prevent Ca2+ influx. A. Representative traces of Ca2+ transients of individual cells by [K+]o and caffeine. B. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted against [K+]o concentration. Values are expressed as mean ± SE (n = 112 for α-RyR and 18 for β-RyR). α-RyR but not β-RyR exhibited Ca2+ transients induced by high [K+]o. C. A representative trace of Ca2+ transients of myotubes expressing α-RyR stimulated with 80 mM [K+]o before and after treatment with 10 µM nifedipine. D. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted with or without 10 µM nifedipine. Values are expressed as mean ± SE (n = 15). Nifedipine inhibited high [K+]o-induced Ca2+ transients.
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pone-0011526-g002: Depolarization-induced Ca2+ release.Intracellular Ca2+ ([Ca2+]i) of 1B5 myotubes transduced with either α-RyR or β-RyR virions was imaged using fluo-4 as described in Materials and Methods. Cells were exposed for 30 sec to varied high [K+]o solutions (with the constant [K+]×[Cl−] product) to trigger DICR, and finally to 20 mM caffeine to confirm the functional expression of RyR. Ca2+ in the bath solution was omitted prior to and during stimuli to prevent Ca2+ influx. A. Representative traces of Ca2+ transients of individual cells by [K+]o and caffeine. B. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted against [K+]o concentration. Values are expressed as mean ± SE (n = 112 for α-RyR and 18 for β-RyR). α-RyR but not β-RyR exhibited Ca2+ transients induced by high [K+]o. C. A representative trace of Ca2+ transients of myotubes expressing α-RyR stimulated with 80 mM [K+]o before and after treatment with 10 µM nifedipine. D. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted with or without 10 µM nifedipine. Values are expressed as mean ± SE (n = 15). Nifedipine inhibited high [K+]o-induced Ca2+ transients.

Mentions: In mock-infected myotubes, neither high [K+]o (up to 80 mM) nor caffeine (up to 20 mM) triggered Ca2+ transients, corresponding to a lack of detected RyRs (data not shown). The efficiency of HSV virion transduction was ∼80%. All myotubes successfully transduced with α-RyR virions exhibited Ca2+ transients induced by increasing [K+]o in a dose-dependent manner and seemed to reach a plateau at 80 mM [K+]o (Fig. 2A, 2B). They also had Ca2+ transients after perfusion with 20 mM caffeine. By contrast, the myotubes transduced with β-RyR responded to caffeine, but no myotubes were responsive to high [K+]o up to 80 mM. Lack of high [K+]o response in β-RyR does not stem from improper expression or targeting of the isoform, because it was expressed as a full-length protein and co-localized with the DHPR (see Fig. 1). The [K+]o dependence of Ca2+ transients by α-RyR demonstrated that the EC50 value of [K+]o was about 28 mM (Fig. 2B), which is in good agreement with the values determined by high [K+]o-induced tension development in intact skeletal muscle fibers [28], [29]. The Increasing [K+]o also accelerated the time-dependent decline in [Ca2+]i which can be explained by inactivation of the voltage sensor (Fig. 2A). The high [K+]o-induced Ca2+ transients were strongly inhibited by 10 µM nifedipine, which selectively blocks the DHPR voltage sensor (Fig. 2C, 2D). This is consistent with the properties of DICR in frog and mammalian skeletal muscles [6], [7] and indicates that frog α-RyR but not β-RyR mediates DICR that is coupled to and controlled by endogenous DHPRs in 1B5 myotubes.


Frog alpha- and beta-ryanodine receptors provide distinct intracellular Ca2+ signals in a myogenic cell line.

Kashiyama T, Murayama T, Suzuki E, Allen PD, Ogawa Y - PLoS ONE (2010)

Depolarization-induced Ca2+ release.Intracellular Ca2+ ([Ca2+]i) of 1B5 myotubes transduced with either α-RyR or β-RyR virions was imaged using fluo-4 as described in Materials and Methods. Cells were exposed for 30 sec to varied high [K+]o solutions (with the constant [K+]×[Cl−] product) to trigger DICR, and finally to 20 mM caffeine to confirm the functional expression of RyR. Ca2+ in the bath solution was omitted prior to and during stimuli to prevent Ca2+ influx. A. Representative traces of Ca2+ transients of individual cells by [K+]o and caffeine. B. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted against [K+]o concentration. Values are expressed as mean ± SE (n = 112 for α-RyR and 18 for β-RyR). α-RyR but not β-RyR exhibited Ca2+ transients induced by high [K+]o. C. A representative trace of Ca2+ transients of myotubes expressing α-RyR stimulated with 80 mM [K+]o before and after treatment with 10 µM nifedipine. D. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted with or without 10 µM nifedipine. Values are expressed as mean ± SE (n = 15). Nifedipine inhibited high [K+]o-induced Ca2+ transients.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2902508&req=5

pone-0011526-g002: Depolarization-induced Ca2+ release.Intracellular Ca2+ ([Ca2+]i) of 1B5 myotubes transduced with either α-RyR or β-RyR virions was imaged using fluo-4 as described in Materials and Methods. Cells were exposed for 30 sec to varied high [K+]o solutions (with the constant [K+]×[Cl−] product) to trigger DICR, and finally to 20 mM caffeine to confirm the functional expression of RyR. Ca2+ in the bath solution was omitted prior to and during stimuli to prevent Ca2+ influx. A. Representative traces of Ca2+ transients of individual cells by [K+]o and caffeine. B. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted against [K+]o concentration. Values are expressed as mean ± SE (n = 112 for α-RyR and 18 for β-RyR). α-RyR but not β-RyR exhibited Ca2+ transients induced by high [K+]o. C. A representative trace of Ca2+ transients of myotubes expressing α-RyR stimulated with 80 mM [K+]o before and after treatment with 10 µM nifedipine. D. Averaged maximal change in fluo-4 fluorescence (ΔF/F0) was plotted with or without 10 µM nifedipine. Values are expressed as mean ± SE (n = 15). Nifedipine inhibited high [K+]o-induced Ca2+ transients.
Mentions: In mock-infected myotubes, neither high [K+]o (up to 80 mM) nor caffeine (up to 20 mM) triggered Ca2+ transients, corresponding to a lack of detected RyRs (data not shown). The efficiency of HSV virion transduction was ∼80%. All myotubes successfully transduced with α-RyR virions exhibited Ca2+ transients induced by increasing [K+]o in a dose-dependent manner and seemed to reach a plateau at 80 mM [K+]o (Fig. 2A, 2B). They also had Ca2+ transients after perfusion with 20 mM caffeine. By contrast, the myotubes transduced with β-RyR responded to caffeine, but no myotubes were responsive to high [K+]o up to 80 mM. Lack of high [K+]o response in β-RyR does not stem from improper expression or targeting of the isoform, because it was expressed as a full-length protein and co-localized with the DHPR (see Fig. 1). The [K+]o dependence of Ca2+ transients by α-RyR demonstrated that the EC50 value of [K+]o was about 28 mM (Fig. 2B), which is in good agreement with the values determined by high [K+]o-induced tension development in intact skeletal muscle fibers [28], [29]. The Increasing [K+]o also accelerated the time-dependent decline in [Ca2+]i which can be explained by inactivation of the voltage sensor (Fig. 2A). The high [K+]o-induced Ca2+ transients were strongly inhibited by 10 µM nifedipine, which selectively blocks the DHPR voltage sensor (Fig. 2C, 2D). This is consistent with the properties of DICR in frog and mammalian skeletal muscles [6], [7] and indicates that frog α-RyR but not β-RyR mediates DICR that is coupled to and controlled by endogenous DHPRs in 1B5 myotubes.

Bottom Line: Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line.These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan.

ABSTRACT

Background: In frog skeletal muscle, two ryanodine receptor (RyR) isoforms, alpha-RyR and beta-RyR, are expressed in nearly equal amounts. However, the roles and significance of the two isoforms in excitation-contraction (E-C) coupling remains to be elucidated.

Methodology/principal findings: In this study, we expressed either or both alpha-RyR and beta-RyR in 1B5 RyR-deficient myotubes using the herpes simplex virus 1 helper-free amplicon system. Immunological characterizations revealed that alpha-RyR and beta-RyR are appropriately expressed and targeted at the junctions in 1B5 myotubes. In Ca(2+) imaging studies, each isoform exhibited caffeine-induced Ca(2+) transients, an indicative of Ca(2+)-induced Ca(2+) release (CICR). However, the fashion of Ca(2+) release events was fundamentally different: alpha-RyR mediated graded and sustained Ca(2+) release observed uniformly throughout the cytoplasm, whereas beta-RyR supported all-or-none type regenerative Ca(2+) oscillations and waves. alpha-RyR but not beta-RyR exhibited Ca(2+) transients triggered by membrane depolarization with high [K(+)](o) that were nifedipine-sensitive, indicating that only alpha-RyR mediates depolarization-induced Ca(2+) release. Myotubes co-expressing alpha-RyR and beta-RyR demonstrated high [K(+)](o)-induced Ca(2+) transients which were indistinguishable from those with myotubes expressing alpha-RyR alone. Furthermore, procaine did not affect the peak height of high [K(+)](o)-induced Ca(2+) transients, suggesting minor amplification of Ca(2+) release by beta-RyR via CICR in 1B5 myotubes.

Conclusions/significance: These findings suggest that alpha-RyR and beta-RyR provide distinct intracellular Ca(2+) signals in a myogenic cell line. These distinct properties may also occur in frog skeletal muscle and will be important for E-C coupling.

Show MeSH
Related in: MedlinePlus