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Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1.

Ito K, Komazaki S, Sasamoto K, Yoshida M, Nishi M, Kitamura K, Takeshima H - J. Cell Biol. (2001)

Bottom Line: Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle.The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+.Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Kurume University and CREST, Japan Science and Technology Corporation, Fukuoka 839-0861, Japan.

ABSTRACT
In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

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Effects of extracellular Ca2+ concentrations on twitch tension in wild-type and JP-1 knockout skeletal muscles. Control twitch tension was measured in modified Krebs-Ringer solution containing 2.5 mM CaCl2. The bathing solution was then changed to a high-Ca2+ solution containing 5 mM CaCl2 (A) or a Ca2+-free solution containing 0.1 mM EGTA (B). Twitches evoked by 0.33-Hz stimuli were monitored continuously after the replacement, and the data (at least n = 6 from four neonates in each plot) were normalized and represent the mean ± SEM. Statistical differences between the genotypes are indicated by asterisks (t test, *P < 0.05 and **P < 0.01).
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fig7: Effects of extracellular Ca2+ concentrations on twitch tension in wild-type and JP-1 knockout skeletal muscles. Control twitch tension was measured in modified Krebs-Ringer solution containing 2.5 mM CaCl2. The bathing solution was then changed to a high-Ca2+ solution containing 5 mM CaCl2 (A) or a Ca2+-free solution containing 0.1 mM EGTA (B). Twitches evoked by 0.33-Hz stimuli were monitored continuously after the replacement, and the data (at least n = 6 from four neonates in each plot) were normalized and represent the mean ± SEM. Statistical differences between the genotypes are indicated by asterisks (t test, *P < 0.05 and **P < 0.01).

Mentions: We then examined the effects of extracellular Ca2+ levels in the JP-1 knockout muscle. In mature muscle, an increase in extracellular Ca2+ does not obviously affect the twitch force, because contraction is produced by Ca2+ release from the SR by functional-coupling DHPR and RyR during E–C coupling. On the other hand, when the extracellular Ca2+ level was increased from 2.5 mM to 5 mM, twitch tension was gradually enhanced in muscle from neonatal mice (Fig. 7 A). The enhancement of twitch amplitude by high Ca2+ in the mutant muscle was significantly higher than that of control muscle. In accordance with a previous report (Beam and Knudson, 1988), the results suggest that immature skeletal muscle cells contain a detectable population of Ca2+ channels that are uncoupled with RyR and devoted to voltage-dependent Ca2+ influx. The highly increased twitch by high Ca2+, together with weak contractile responses at low-frequency stimuli (Fig. 6), might suggest an increased population of Ca2+ channels uncoupled with RyR in the JP-1 knockout muscle. Alternatively, the abnormal response to extracellular Ca2+ might require dysregulation of SR Ca2+ loading in the mutant muscle.


Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1.

Ito K, Komazaki S, Sasamoto K, Yoshida M, Nishi M, Kitamura K, Takeshima H - J. Cell Biol. (2001)

Effects of extracellular Ca2+ concentrations on twitch tension in wild-type and JP-1 knockout skeletal muscles. Control twitch tension was measured in modified Krebs-Ringer solution containing 2.5 mM CaCl2. The bathing solution was then changed to a high-Ca2+ solution containing 5 mM CaCl2 (A) or a Ca2+-free solution containing 0.1 mM EGTA (B). Twitches evoked by 0.33-Hz stimuli were monitored continuously after the replacement, and the data (at least n = 6 from four neonates in each plot) were normalized and represent the mean ± SEM. Statistical differences between the genotypes are indicated by asterisks (t test, *P < 0.05 and **P < 0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196186&req=5

fig7: Effects of extracellular Ca2+ concentrations on twitch tension in wild-type and JP-1 knockout skeletal muscles. Control twitch tension was measured in modified Krebs-Ringer solution containing 2.5 mM CaCl2. The bathing solution was then changed to a high-Ca2+ solution containing 5 mM CaCl2 (A) or a Ca2+-free solution containing 0.1 mM EGTA (B). Twitches evoked by 0.33-Hz stimuli were monitored continuously after the replacement, and the data (at least n = 6 from four neonates in each plot) were normalized and represent the mean ± SEM. Statistical differences between the genotypes are indicated by asterisks (t test, *P < 0.05 and **P < 0.01).
Mentions: We then examined the effects of extracellular Ca2+ levels in the JP-1 knockout muscle. In mature muscle, an increase in extracellular Ca2+ does not obviously affect the twitch force, because contraction is produced by Ca2+ release from the SR by functional-coupling DHPR and RyR during E–C coupling. On the other hand, when the extracellular Ca2+ level was increased from 2.5 mM to 5 mM, twitch tension was gradually enhanced in muscle from neonatal mice (Fig. 7 A). The enhancement of twitch amplitude by high Ca2+ in the mutant muscle was significantly higher than that of control muscle. In accordance with a previous report (Beam and Knudson, 1988), the results suggest that immature skeletal muscle cells contain a detectable population of Ca2+ channels that are uncoupled with RyR and devoted to voltage-dependent Ca2+ influx. The highly increased twitch by high Ca2+, together with weak contractile responses at low-frequency stimuli (Fig. 6), might suggest an increased population of Ca2+ channels uncoupled with RyR in the JP-1 knockout muscle. Alternatively, the abnormal response to extracellular Ca2+ might require dysregulation of SR Ca2+ loading in the mutant muscle.

Bottom Line: Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle.The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+.Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, Kurume University and CREST, Japan Science and Technology Corporation, Fukuoka 839-0861, Japan.

ABSTRACT
In skeletal muscle excitation-contraction (E-C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E-C coupling in skeletal muscle.

Show MeSH
Related in: MedlinePlus