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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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Generation of JP-1 knockout mice. (A) Homologous recombination at the mouse JP-1 locus. Restriction enzyme maps of the wild-type allele, targeting vector, and mutant allele are illustrated. The exons in the gene (E1 and E2) are indicated by filled boxes, and the neomycin resistance gene (neo) and diphtheria toxin gene (DTA) are indicated by open boxes; the directions of transcription are shown by arrows. The hybridization probe and PCR primers for detection of the mutant gene are indicated by a hatched box and opened arrows, respectively; the predicted sizes of the DNA fragments in Southern blot analysis and PCR are also shown. (B) Detection of mutant JP-1 allele by PCR. Genomic DNAs from newborn mice were analyzed by PCR using primers indicated in A. Amplified DNA fragments were run on a 1.5% agarose gel, and size markers are indicated in base pairs. Positions of PCR products from wild-type and mutant genes are given. Immunoblot analysis of JP-1 (C) and JP-2 (D) in newborn mice. Microsomal proteins from hindlimb preparations were analyzed using specific antibody against JP-1 in C or JP-2 in D. Positions for target proteins are given, and size markers are indicated in kilodaltons. (E) Immunofluorescence detection of JP-2 in JP-1 knockout muscle. Cryosections of hindlimb muscles from wild-type and JP-1 knockout neonates were examined using an antibody specific to JP-2. No difference in staining pattern was detected between the genotypes. The partial sequence data for the JP-1 gene has been deposited in the database (GenBank/EMBL/DDBJ accession no. AB024446). Bar, 3 μm.
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fig2: Generation of JP-1 knockout mice. (A) Homologous recombination at the mouse JP-1 locus. Restriction enzyme maps of the wild-type allele, targeting vector, and mutant allele are illustrated. The exons in the gene (E1 and E2) are indicated by filled boxes, and the neomycin resistance gene (neo) and diphtheria toxin gene (DTA) are indicated by open boxes; the directions of transcription are shown by arrows. The hybridization probe and PCR primers for detection of the mutant gene are indicated by a hatched box and opened arrows, respectively; the predicted sizes of the DNA fragments in Southern blot analysis and PCR are also shown. (B) Detection of mutant JP-1 allele by PCR. Genomic DNAs from newborn mice were analyzed by PCR using primers indicated in A. Amplified DNA fragments were run on a 1.5% agarose gel, and size markers are indicated in base pairs. Positions of PCR products from wild-type and mutant genes are given. Immunoblot analysis of JP-1 (C) and JP-2 (D) in newborn mice. Microsomal proteins from hindlimb preparations were analyzed using specific antibody against JP-1 in C or JP-2 in D. Positions for target proteins are given, and size markers are indicated in kilodaltons. (E) Immunofluorescence detection of JP-2 in JP-1 knockout muscle. Cryosections of hindlimb muscles from wild-type and JP-1 knockout neonates were examined using an antibody specific to JP-2. No difference in staining pattern was detected between the genotypes. The partial sequence data for the JP-1 gene has been deposited in the database (GenBank/EMBL/DDBJ accession no. AB024446). Bar, 3 μm.

Mentions: 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.


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)

Generation of JP-1 knockout mice. (A) Homologous recombination at the mouse JP-1 locus. Restriction enzyme maps of the wild-type allele, targeting vector, and mutant allele are illustrated. The exons in the gene (E1 and E2) are indicated by filled boxes, and the neomycin resistance gene (neo) and diphtheria toxin gene (DTA) are indicated by open boxes; the directions of transcription are shown by arrows. The hybridization probe and PCR primers for detection of the mutant gene are indicated by a hatched box and opened arrows, respectively; the predicted sizes of the DNA fragments in Southern blot analysis and PCR are also shown. (B) Detection of mutant JP-1 allele by PCR. Genomic DNAs from newborn mice were analyzed by PCR using primers indicated in A. Amplified DNA fragments were run on a 1.5% agarose gel, and size markers are indicated in base pairs. Positions of PCR products from wild-type and mutant genes are given. Immunoblot analysis of JP-1 (C) and JP-2 (D) in newborn mice. Microsomal proteins from hindlimb preparations were analyzed using specific antibody against JP-1 in C or JP-2 in D. Positions for target proteins are given, and size markers are indicated in kilodaltons. (E) Immunofluorescence detection of JP-2 in JP-1 knockout muscle. Cryosections of hindlimb muscles from wild-type and JP-1 knockout neonates were examined using an antibody specific to JP-2. No difference in staining pattern was detected between the genotypes. The partial sequence data for the JP-1 gene has been deposited in the database (GenBank/EMBL/DDBJ accession no. AB024446). Bar, 3 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196186&req=5

fig2: Generation of JP-1 knockout mice. (A) Homologous recombination at the mouse JP-1 locus. Restriction enzyme maps of the wild-type allele, targeting vector, and mutant allele are illustrated. The exons in the gene (E1 and E2) are indicated by filled boxes, and the neomycin resistance gene (neo) and diphtheria toxin gene (DTA) are indicated by open boxes; the directions of transcription are shown by arrows. The hybridization probe and PCR primers for detection of the mutant gene are indicated by a hatched box and opened arrows, respectively; the predicted sizes of the DNA fragments in Southern blot analysis and PCR are also shown. (B) Detection of mutant JP-1 allele by PCR. Genomic DNAs from newborn mice were analyzed by PCR using primers indicated in A. Amplified DNA fragments were run on a 1.5% agarose gel, and size markers are indicated in base pairs. Positions of PCR products from wild-type and mutant genes are given. Immunoblot analysis of JP-1 (C) and JP-2 (D) in newborn mice. Microsomal proteins from hindlimb preparations were analyzed using specific antibody against JP-1 in C or JP-2 in D. Positions for target proteins are given, and size markers are indicated in kilodaltons. (E) Immunofluorescence detection of JP-2 in JP-1 knockout muscle. Cryosections of hindlimb muscles from wild-type and JP-1 knockout neonates were examined using an antibody specific to JP-2. No difference in staining pattern was detected between the genotypes. The partial sequence data for the JP-1 gene has been deposited in the database (GenBank/EMBL/DDBJ accession no. AB024446). Bar, 3 μm.
Mentions: 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.

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