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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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Spatiotemporal properties of Ca2+ release.A, B. Line scan analysis of Ca2+ transients of 1B5 myotubes expressing α-RyR (A) and β-RyR (B) induced by caffeine or high [K+]o. Scan lines were set on along their longitudinal axes of myotubes. Ca2+ transients within myotubes expressing α-RyR uniformly occurred instantaneously throughout the cells, whereas those of myotubes expressing β-RyR occurred at specific sites and propagated in waves within the cell. C, D. Wave velocity of individual cells expressing β-RyR (C) and its average (D) was plotted against caffeine dose. Wave velocity increased with caffeine dose (mean±SE, n = 15).
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pone-0011526-g004: Spatiotemporal properties of Ca2+ release.A, B. Line scan analysis of Ca2+ transients of 1B5 myotubes expressing α-RyR (A) and β-RyR (B) induced by caffeine or high [K+]o. Scan lines were set on along their longitudinal axes of myotubes. Ca2+ transients within myotubes expressing α-RyR uniformly occurred instantaneously throughout the cells, whereas those of myotubes expressing β-RyR occurred at specific sites and propagated in waves within the cell. C, D. Wave velocity of individual cells expressing β-RyR (C) and its average (D) was plotted against caffeine dose. Wave velocity increased with caffeine dose (mean±SE, n = 15).

Mentions: The above results suggest a substantial difference in Ca2+ release properties of α-RyR and β-RyR. To deepen understanding of the Ca2+ release mechanism, we investigated spatiotemporal properties of Ca2+ transients of the two RyR isoforms. Caffeine-induced Ca2+ transients in myotubes expressing α-RyR occurred uniformly within the cell (Movie S1). Line scan analysis along their longitudinal axes of the myotubes clearly demonstrates a uniform increase in Ca2+ throughout the cell (Fig. 4A). Similar responses were also observed when myotubes were stimulated with high [K+]o (Fig. 4A, Movie S2). In contrast, myotubes expressing β-RyR exhibited Ca2+ waves, which occurred at specific sites and propagated within cells (Fig. 4B, Movie S3). At a higher caffeine doses in cells expressing β-RyR (≥5 mM), the initial Ca2+ transients occurred almost instantaneously throughout the myotubes, followed by the repetitive Ca2+ waves. Notably, velocity of the Ca2+ waves increased with caffeine dose, although substantial cell-to-cell variations were apparent (Figs. 4C). Averaged velocities of Ca2+ waves in myotubes expressing β-RyR were 15±3 µm/sec at 0.2 mM caffeine, 18±2 µm/sec at 0.5 mM caffeine, 30±4 µm/sec at 1 mM caffeine, 44±5 µm/sec at 2 mM caffeine, and 72±9 µm/sec at 5 mM caffeine (Fig. 4D). The velocities are similar with those of the caffeine-induced Ca2+ waves of frog skeletal muscle [30] as well as the other cell types [31], [32]. These findings suggest that α-RyR and β-RyR have distinct spatiotemporal properties of Ca2+ release 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)

Spatiotemporal properties of Ca2+ release.A, B. Line scan analysis of Ca2+ transients of 1B5 myotubes expressing α-RyR (A) and β-RyR (B) induced by caffeine or high [K+]o. Scan lines were set on along their longitudinal axes of myotubes. Ca2+ transients within myotubes expressing α-RyR uniformly occurred instantaneously throughout the cells, whereas those of myotubes expressing β-RyR occurred at specific sites and propagated in waves within the cell. C, D. Wave velocity of individual cells expressing β-RyR (C) and its average (D) was plotted against caffeine dose. Wave velocity increased with caffeine dose (mean±SE, n = 15).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011526-g004: Spatiotemporal properties of Ca2+ release.A, B. Line scan analysis of Ca2+ transients of 1B5 myotubes expressing α-RyR (A) and β-RyR (B) induced by caffeine or high [K+]o. Scan lines were set on along their longitudinal axes of myotubes. Ca2+ transients within myotubes expressing α-RyR uniformly occurred instantaneously throughout the cells, whereas those of myotubes expressing β-RyR occurred at specific sites and propagated in waves within the cell. C, D. Wave velocity of individual cells expressing β-RyR (C) and its average (D) was plotted against caffeine dose. Wave velocity increased with caffeine dose (mean±SE, n = 15).
Mentions: The above results suggest a substantial difference in Ca2+ release properties of α-RyR and β-RyR. To deepen understanding of the Ca2+ release mechanism, we investigated spatiotemporal properties of Ca2+ transients of the two RyR isoforms. Caffeine-induced Ca2+ transients in myotubes expressing α-RyR occurred uniformly within the cell (Movie S1). Line scan analysis along their longitudinal axes of the myotubes clearly demonstrates a uniform increase in Ca2+ throughout the cell (Fig. 4A). Similar responses were also observed when myotubes were stimulated with high [K+]o (Fig. 4A, Movie S2). In contrast, myotubes expressing β-RyR exhibited Ca2+ waves, which occurred at specific sites and propagated within cells (Fig. 4B, Movie S3). At a higher caffeine doses in cells expressing β-RyR (≥5 mM), the initial Ca2+ transients occurred almost instantaneously throughout the myotubes, followed by the repetitive Ca2+ waves. Notably, velocity of the Ca2+ waves increased with caffeine dose, although substantial cell-to-cell variations were apparent (Figs. 4C). Averaged velocities of Ca2+ waves in myotubes expressing β-RyR were 15±3 µm/sec at 0.2 mM caffeine, 18±2 µm/sec at 0.5 mM caffeine, 30±4 µm/sec at 1 mM caffeine, 44±5 µm/sec at 2 mM caffeine, and 72±9 µm/sec at 5 mM caffeine (Fig. 4D). The velocities are similar with those of the caffeine-induced Ca2+ waves of frog skeletal muscle [30] as well as the other cell types [31], [32]. These findings suggest that α-RyR and β-RyR have distinct spatiotemporal properties of Ca2+ release 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