Limits...
Adult onset cardiac dilatation in a transgenic mouse line with Galβ1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy.

Suzuki O, Kanai T, Nishikawa T, Yamamoto Y, Noguchi A, Takimoto K, Koura M, Noguchi Y, Uchio-Yamada K, Tsuji S, Matsuda J - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

Bottom Line: Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases.Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered.Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Models for Human Diseases, National Institute of Biomedical Innovation, Ibaraki, Japan. osuzuki@nibio.go.jp

ABSTRACT
Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Galβ1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.

Show MeSH

Related in: MedlinePlus

Production of ST3Gal-II transgenic mice. (A) SalI-BamHI fragments of a pCAGGS-based plasmid containing mouse ST3Gal-II cDNA were injected into mouse zygotes. (B) Integration of the transgene was confirmed by Southern blot analysis. Lane 1: transgenic mice, Lane 2: non-transgenic (WT) mice, Lane 3: C57BL/6Cr. M: DNA size markers. (C) Expression of the transgene was confirmed by TaqMan based quantitative RT-PCR analysis of hearts from homozygous (n = 5), hemizygous (n = 3), and wild (n = 5) mice at 3 months of age. ST3Gal-II expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. Values with different labels (a, b, and c) are significantly different (p < 0.05). (D) Western blot analysis indicated significantly higher expression (*, p < 0.05) of ST3Gal-II protein in homozygous TG hearts than WT hearts at 10 weeks of age (n = 3). (E) Our previous study14) indicated that the transgene was inserted at ∼200 kb upstream of the SGCD gene. (F) No significant difference in the SGCD expressions in hearts or skeletal muscles (rectus femoris) between TG (n = 4, black bars) and WT (n = 5, white bars) was found by TaqMan based quantitative RT-PCR analysis of 3-month-old mice. SGCD expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. (G) Western blot analysis indicated no significant difference in expression of δ-sarcoglycan protein between TG and WT hearts at 10 weeks of age (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3313694&req=5

fig01: Production of ST3Gal-II transgenic mice. (A) SalI-BamHI fragments of a pCAGGS-based plasmid containing mouse ST3Gal-II cDNA were injected into mouse zygotes. (B) Integration of the transgene was confirmed by Southern blot analysis. Lane 1: transgenic mice, Lane 2: non-transgenic (WT) mice, Lane 3: C57BL/6Cr. M: DNA size markers. (C) Expression of the transgene was confirmed by TaqMan based quantitative RT-PCR analysis of hearts from homozygous (n = 5), hemizygous (n = 3), and wild (n = 5) mice at 3 months of age. ST3Gal-II expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. Values with different labels (a, b, and c) are significantly different (p < 0.05). (D) Western blot analysis indicated significantly higher expression (*, p < 0.05) of ST3Gal-II protein in homozygous TG hearts than WT hearts at 10 weeks of age (n = 3). (E) Our previous study14) indicated that the transgene was inserted at ∼200 kb upstream of the SGCD gene. (F) No significant difference in the SGCD expressions in hearts or skeletal muscles (rectus femoris) between TG (n = 4, black bars) and WT (n = 5, white bars) was found by TaqMan based quantitative RT-PCR analysis of 3-month-old mice. SGCD expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. (G) Western blot analysis indicated no significant difference in expression of δ-sarcoglycan protein between TG and WT hearts at 10 weeks of age (n = 3).

Mentions: The mouse ST3Gal-II cDNA sequence (GenBank: X76989) was cloned in our previous study.3) The overexpression construct was generated by inserting the cDNA sequence into the multicloning site of a pCAGGS-based plasmid.13) The SalI-BamHI fragments of the plasmid (Fig. 1A) were injected into the pronuclei of C57BL/6CrSlc zygotes. The manipulated embryos were transferred into the oviducts of pseudopregnant Slc:ICR mice. Tail DNA of transgenic founder mice was first screened by PCR. Integration of the transgene into founder candidates was assessed by Southern blot analysis with EcoRI-digested genomic DNA (3 µg/lane) from 4-week-old mice (probe 1: SalI-SmaBI fragment of the CAGGS plasmid, 357 bp; probe 2: KpnI-XhoI fragment of ST3Gal-II cDNA, 930 bp; Fig. 1A). In our previous study,14) the insertion site of the transgene was mapped by Blast search for the flanking sequence of the transgene in the Ensembl database (http://www.ensembl.org/, Fig. 1E).


Adult onset cardiac dilatation in a transgenic mouse line with Galβ1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes: a new model for dilated cardiomyopathy.

Suzuki O, Kanai T, Nishikawa T, Yamamoto Y, Noguchi A, Takimoto K, Koura M, Noguchi Y, Uchio-Yamada K, Tsuji S, Matsuda J - Proc. Jpn. Acad., Ser. B, Phys. Biol. Sci. (2011)

Production of ST3Gal-II transgenic mice. (A) SalI-BamHI fragments of a pCAGGS-based plasmid containing mouse ST3Gal-II cDNA were injected into mouse zygotes. (B) Integration of the transgene was confirmed by Southern blot analysis. Lane 1: transgenic mice, Lane 2: non-transgenic (WT) mice, Lane 3: C57BL/6Cr. M: DNA size markers. (C) Expression of the transgene was confirmed by TaqMan based quantitative RT-PCR analysis of hearts from homozygous (n = 5), hemizygous (n = 3), and wild (n = 5) mice at 3 months of age. ST3Gal-II expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. Values with different labels (a, b, and c) are significantly different (p < 0.05). (D) Western blot analysis indicated significantly higher expression (*, p < 0.05) of ST3Gal-II protein in homozygous TG hearts than WT hearts at 10 weeks of age (n = 3). (E) Our previous study14) indicated that the transgene was inserted at ∼200 kb upstream of the SGCD gene. (F) No significant difference in the SGCD expressions in hearts or skeletal muscles (rectus femoris) between TG (n = 4, black bars) and WT (n = 5, white bars) was found by TaqMan based quantitative RT-PCR analysis of 3-month-old mice. SGCD expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. (G) Western blot analysis indicated no significant difference in expression of δ-sarcoglycan protein between TG and WT hearts at 10 weeks of age (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3313694&req=5

fig01: Production of ST3Gal-II transgenic mice. (A) SalI-BamHI fragments of a pCAGGS-based plasmid containing mouse ST3Gal-II cDNA were injected into mouse zygotes. (B) Integration of the transgene was confirmed by Southern blot analysis. Lane 1: transgenic mice, Lane 2: non-transgenic (WT) mice, Lane 3: C57BL/6Cr. M: DNA size markers. (C) Expression of the transgene was confirmed by TaqMan based quantitative RT-PCR analysis of hearts from homozygous (n = 5), hemizygous (n = 3), and wild (n = 5) mice at 3 months of age. ST3Gal-II expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. Values with different labels (a, b, and c) are significantly different (p < 0.05). (D) Western blot analysis indicated significantly higher expression (*, p < 0.05) of ST3Gal-II protein in homozygous TG hearts than WT hearts at 10 weeks of age (n = 3). (E) Our previous study14) indicated that the transgene was inserted at ∼200 kb upstream of the SGCD gene. (F) No significant difference in the SGCD expressions in hearts or skeletal muscles (rectus femoris) between TG (n = 4, black bars) and WT (n = 5, white bars) was found by TaqMan based quantitative RT-PCR analysis of 3-month-old mice. SGCD expression was normalized with GAPDH expression and displayed as mean ± S.D. with the average of wild values as 1. (G) Western blot analysis indicated no significant difference in expression of δ-sarcoglycan protein between TG and WT hearts at 10 weeks of age (n = 3).
Mentions: The mouse ST3Gal-II cDNA sequence (GenBank: X76989) was cloned in our previous study.3) The overexpression construct was generated by inserting the cDNA sequence into the multicloning site of a pCAGGS-based plasmid.13) The SalI-BamHI fragments of the plasmid (Fig. 1A) were injected into the pronuclei of C57BL/6CrSlc zygotes. The manipulated embryos were transferred into the oviducts of pseudopregnant Slc:ICR mice. Tail DNA of transgenic founder mice was first screened by PCR. Integration of the transgene into founder candidates was assessed by Southern blot analysis with EcoRI-digested genomic DNA (3 µg/lane) from 4-week-old mice (probe 1: SalI-SmaBI fragment of the CAGGS plasmid, 357 bp; probe 2: KpnI-XhoI fragment of ST3Gal-II cDNA, 930 bp; Fig. 1A). In our previous study,14) the insertion site of the transgene was mapped by Blast search for the flanking sequence of the transgene in the Ensembl database (http://www.ensembl.org/, Fig. 1E).

Bottom Line: Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases.Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered.Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Animal Models for Human Diseases, National Institute of Biomedical Innovation, Ibaraki, Japan. osuzuki@nibio.go.jp

ABSTRACT
Sugar chain abnormalities in glycolipids and glycoproteins are associated with various diseases. Here, we report an adult onset cardiac dilatation in a transgenic mouse line with Galβ1,3GalNAc α2,3-sialyltransferase II (ST3Gal-II) transgenes. The transgenic hearts at the end-stage, at around 7 months old, were enlarged, with enlarged cavities and thin, low-tensile walls, typical of dilated cardiomyopathy. Although no apparent change was found in heart gangliosides, glycosylation of heart proteins was altered. Interestingly, sugar moieties not directly related to the ST3Gal-II catalytic reaction were also changed. Significant increases in calreticulin and calnexin were observed in hearts of the transgenic mice. These results suggest that expression of ST3Gal-II transgenes induces abnormal protein glycosylation, which disorganizes the endoplasmic/sarcoplasmic reticulum quality control system and elevates the calreticulin/calnexin level, resulting in suppression of cardiac function. The transgenic mice showed 100% incidence of adult onset cardiac dilatation, suggesting great potential as a new model for dilated cardiomyopathy.

Show MeSH
Related in: MedlinePlus