Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of JP subtypes in skeletal muscle. (A) Western blot analysis of JP subtypes in mouse tissues. Total microsomes from adult mouse tissues (15 μg protein each) were analyzed with antibodies specific to JP-1 and JP-2. B, brain; H, heart; K, kidney; L, liver; SM, skeletal muscle. Size markers are shown in kilodaltons. JP-2 was detected as a broad band in skeletal muscle due to comigration with Ca2+-ATPase, the major protein component in the SR. (B) Western blot analysis of JP subtypes during muscle maturation. Total hindlimb microsomes (40 μg protein each) prepared from embryonic day 14 (E14) to postnatal day 28 (P28) mice were analyzed using the subtype-specific antibodies. Although expression of JP-1 in embryos and neonates could be detected in longer exposure (see Fig. 2), the signal densities were markedly lower than those of young adult mice. In contrast with JP-1, induction of JP-2 expression was relatively loose during muscle maturation. (C) Immunohistochemical analysis of JP subtypes in skeletal muscle. A cryosection of hindlimb muscle from adult mouse was labeled by immunofluorescence using antibodies specific to JP-1 (on 543 nm excitation) and JP-2 (on 488 nm excitation). Cytoplasmic rows immunolabeled with both antibodies are localized in identical positions (Merged). Essentially the same staining patterns were observed in all muscle fibers examined in hindlimb muscle. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196186&req=5

fig1: Expression of JP subtypes in skeletal muscle. (A) Western blot analysis of JP subtypes in mouse tissues. Total microsomes from adult mouse tissues (15 μg protein each) were analyzed with antibodies specific to JP-1 and JP-2. B, brain; H, heart; K, kidney; L, liver; SM, skeletal muscle. Size markers are shown in kilodaltons. JP-2 was detected as a broad band in skeletal muscle due to comigration with Ca2+-ATPase, the major protein component in the SR. (B) Western blot analysis of JP subtypes during muscle maturation. Total hindlimb microsomes (40 μg protein each) prepared from embryonic day 14 (E14) to postnatal day 28 (P28) mice were analyzed using the subtype-specific antibodies. Although expression of JP-1 in embryos and neonates could be detected in longer exposure (see Fig. 2), the signal densities were markedly lower than those of young adult mice. In contrast with JP-1, induction of JP-2 expression was relatively loose during muscle maturation. (C) Immunohistochemical analysis of JP subtypes in skeletal muscle. A cryosection of hindlimb muscle from adult mouse was labeled by immunofluorescence using antibodies specific to JP-1 (on 543 nm excitation) and JP-2 (on 488 nm excitation). Cytoplasmic rows immunolabeled with both antibodies are localized in identical positions (Merged). Essentially the same staining patterns were observed in all muscle fibers examined in hindlimb muscle. Bar, 10 μm.

Mentions: Our results in RNA blot hybridization have suggested that both JP-1 and JP-2 are expressed in skeletal muscle cells (Takeshima et al., 2000). To further examine the expression, we prepared bacterial fusion proteins carrying the divergent regions of JPs, in which no conserved amino acid sequences are observed among the subtypes, and subtype-specific antibodies were developed using the fusion proteins as antigens. In immunoblot analysis of microsomal preparations from adult mouse tissues, the resulting antibodies were subtype specific, and JP-1 and JP-2 were detected as protein bands showing distinguishable migrations on gel electrophoresis (Fig. 1 A). JP-1 was specifically detected in skeletal muscle, and JP-2 was found in both skeletal and cardiac muscles. The data obtained in the Northern and Western analyses were consistent. The analysis in mouse hindlimb muscle showed that the expression levels of both JP-1 and JP-2 significantly increase during muscle maturation (Fig. 1 B). The induction of JP-1 during muscle development is especially remarkable in that the expression levels were very low in embryos and neonates, but muscles from young adult mice contained abundant JP-1. Histochemical analysis demonstrated that both antibodies to JP-1 and JP-2 specifically reacted with the rows localized at A-I junctions in the longitudinal sections of muscle preparations (Fig. 1 C). Because all muscle cells examined in hindlimb were immunolabeled with both antibodies, the JP subtypes are coexpressed in both fast and slow fibers. Our previous results demonstrated specific localization of JP-1 in the skeletal muscle triad junction (Takeshima et al., 2000). The present results showed that both JP-1 and JP-2 are colocalized in the triad junction.


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)

Expression of JP subtypes in skeletal muscle. (A) Western blot analysis of JP subtypes in mouse tissues. Total microsomes from adult mouse tissues (15 μg protein each) were analyzed with antibodies specific to JP-1 and JP-2. B, brain; H, heart; K, kidney; L, liver; SM, skeletal muscle. Size markers are shown in kilodaltons. JP-2 was detected as a broad band in skeletal muscle due to comigration with Ca2+-ATPase, the major protein component in the SR. (B) Western blot analysis of JP subtypes during muscle maturation. Total hindlimb microsomes (40 μg protein each) prepared from embryonic day 14 (E14) to postnatal day 28 (P28) mice were analyzed using the subtype-specific antibodies. Although expression of JP-1 in embryos and neonates could be detected in longer exposure (see Fig. 2), the signal densities were markedly lower than those of young adult mice. In contrast with JP-1, induction of JP-2 expression was relatively loose during muscle maturation. (C) Immunohistochemical analysis of JP subtypes in skeletal muscle. A cryosection of hindlimb muscle from adult mouse was labeled by immunofluorescence using antibodies specific to JP-1 (on 543 nm excitation) and JP-2 (on 488 nm excitation). Cytoplasmic rows immunolabeled with both antibodies are localized in identical positions (Merged). Essentially the same staining patterns were observed in all muscle fibers examined in hindlimb muscle. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Expression of JP subtypes in skeletal muscle. (A) Western blot analysis of JP subtypes in mouse tissues. Total microsomes from adult mouse tissues (15 μg protein each) were analyzed with antibodies specific to JP-1 and JP-2. B, brain; H, heart; K, kidney; L, liver; SM, skeletal muscle. Size markers are shown in kilodaltons. JP-2 was detected as a broad band in skeletal muscle due to comigration with Ca2+-ATPase, the major protein component in the SR. (B) Western blot analysis of JP subtypes during muscle maturation. Total hindlimb microsomes (40 μg protein each) prepared from embryonic day 14 (E14) to postnatal day 28 (P28) mice were analyzed using the subtype-specific antibodies. Although expression of JP-1 in embryos and neonates could be detected in longer exposure (see Fig. 2), the signal densities were markedly lower than those of young adult mice. In contrast with JP-1, induction of JP-2 expression was relatively loose during muscle maturation. (C) Immunohistochemical analysis of JP subtypes in skeletal muscle. A cryosection of hindlimb muscle from adult mouse was labeled by immunofluorescence using antibodies specific to JP-1 (on 543 nm excitation) and JP-2 (on 488 nm excitation). Cytoplasmic rows immunolabeled with both antibodies are localized in identical positions (Merged). Essentially the same staining patterns were observed in all muscle fibers examined in hindlimb muscle. Bar, 10 μm.
Mentions: Our results in RNA blot hybridization have suggested that both JP-1 and JP-2 are expressed in skeletal muscle cells (Takeshima et al., 2000). To further examine the expression, we prepared bacterial fusion proteins carrying the divergent regions of JPs, in which no conserved amino acid sequences are observed among the subtypes, and subtype-specific antibodies were developed using the fusion proteins as antigens. In immunoblot analysis of microsomal preparations from adult mouse tissues, the resulting antibodies were subtype specific, and JP-1 and JP-2 were detected as protein bands showing distinguishable migrations on gel electrophoresis (Fig. 1 A). JP-1 was specifically detected in skeletal muscle, and JP-2 was found in both skeletal and cardiac muscles. The data obtained in the Northern and Western analyses were consistent. The analysis in mouse hindlimb muscle showed that the expression levels of both JP-1 and JP-2 significantly increase during muscle maturation (Fig. 1 B). The induction of JP-1 during muscle development is especially remarkable in that the expression levels were very low in embryos and neonates, but muscles from young adult mice contained abundant JP-1. Histochemical analysis demonstrated that both antibodies to JP-1 and JP-2 specifically reacted with the rows localized at A-I junctions in the longitudinal sections of muscle preparations (Fig. 1 C). Because all muscle cells examined in hindlimb were immunolabeled with both antibodies, the JP subtypes are coexpressed in both fast and slow fibers. Our previous results demonstrated specific localization of JP-1 in the skeletal muscle triad junction (Takeshima et al., 2000). The present results showed that both JP-1 and JP-2 are colocalized in the triad junction.

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