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Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.

Krusche CA, Holthöfer B, Hofe V, van de Sandt AM, Eshkind L, Bockamp E, Merx MW, Kant S, Windoffer R, Leube RE - Basic Res. Cardiol. (2011)

Bottom Line: Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed.Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue.Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Wendlingweg 2, Aachen, Germany. ckrusche@ukaachen.de

ABSTRACT
Desmosomes are cell-cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.

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Preparation of DSG2-mutant mice. a An EcoRV (EV)-segment of the DSG2 wild-type allele (DSG2wt) extending between introns 1 and 8 is depicted at the top. Homologous recombination within this segment leads to the DSG2loxP-neo allele including loxP sites in introns 3 and 6 and an FRT-flanked neomycin-resistance cassette (NFL) in intron 6. Flp-mediated excision of NFL results in allele DSG2loxP. Further recombination by Cre-recombinase generates the mutant allele DSG2mt. Gene regions corresponding to the sequences provided by the targeting construct are indicated by solid lines, and adjoining endogenous gene regions by dotted lines. b Scheme depicting wild-type and mutant Dsg2 (Dsg2wt, Dsg2mt). Dsg2mt lacks parts of the extracellular EC1 and EC2 domains. The scheme further depicts the aminoterminal precursor-specific segment P, the four ~110 amino acid-long extracellular calcium-binding domains EC1–EC4, the extracellular anchor domain EA, transmembrane domain TM, intracellular anchor domain IA, intracellular catenin-binding and cadherin-like sequence ICS, proline-rich linker L, the repeated unit domain RUD and the carboxyterminal tail T. c Detection of mutated DSG2 mRNA. RNA was prepared from DSG2 wild-type and mutant mice (DSG2wt, DSG2mt). GAPDH mRNA was amplified as control. d Analysis of Dsg2 expression by immunoblotting shows a protein band at ~160 kDa in the DSG2wt/wt and of ~110 kDa in DSG2mt/mt mice. The significant difference in mobility is due to deletion of the single glycosylation site. The calculated molecular weight for wild-type Dsg2 is 122,398 and for mutant Dsg2 104,703. Desmocollin 2 (Dsc2), desmoplakin (Dsp) and β-actin served as loading controls. e Immunohistochemistry reveals correct targeting of Dsg2mt to intercalated discs (arrowheads), although expression levels are reduced in DSG2mt/mt mice (same magnification in all images; scale bar, 20 μm). The quantification was done for four wild-type and four mutant 2-week-old littermates from two litters analyzing the ratio of Dsg2 to desmoplakin fluorescence for 40 intercalated discs in each animal
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Fig1: Preparation of DSG2-mutant mice. a An EcoRV (EV)-segment of the DSG2 wild-type allele (DSG2wt) extending between introns 1 and 8 is depicted at the top. Homologous recombination within this segment leads to the DSG2loxP-neo allele including loxP sites in introns 3 and 6 and an FRT-flanked neomycin-resistance cassette (NFL) in intron 6. Flp-mediated excision of NFL results in allele DSG2loxP. Further recombination by Cre-recombinase generates the mutant allele DSG2mt. Gene regions corresponding to the sequences provided by the targeting construct are indicated by solid lines, and adjoining endogenous gene regions by dotted lines. b Scheme depicting wild-type and mutant Dsg2 (Dsg2wt, Dsg2mt). Dsg2mt lacks parts of the extracellular EC1 and EC2 domains. The scheme further depicts the aminoterminal precursor-specific segment P, the four ~110 amino acid-long extracellular calcium-binding domains EC1–EC4, the extracellular anchor domain EA, transmembrane domain TM, intracellular anchor domain IA, intracellular catenin-binding and cadherin-like sequence ICS, proline-rich linker L, the repeated unit domain RUD and the carboxyterminal tail T. c Detection of mutated DSG2 mRNA. RNA was prepared from DSG2 wild-type and mutant mice (DSG2wt, DSG2mt). GAPDH mRNA was amplified as control. d Analysis of Dsg2 expression by immunoblotting shows a protein band at ~160 kDa in the DSG2wt/wt and of ~110 kDa in DSG2mt/mt mice. The significant difference in mobility is due to deletion of the single glycosylation site. The calculated molecular weight for wild-type Dsg2 is 122,398 and for mutant Dsg2 104,703. Desmocollin 2 (Dsc2), desmoplakin (Dsp) and β-actin served as loading controls. e Immunohistochemistry reveals correct targeting of Dsg2mt to intercalated discs (arrowheads), although expression levels are reduced in DSG2mt/mt mice (same magnification in all images; scale bar, 20 μm). The quantification was done for four wild-type and four mutant 2-week-old littermates from two litters analyzing the ratio of Dsg2 to desmoplakin fluorescence for 40 intercalated discs in each animal

Mentions: A targeting construct was prepared for inducible deletion of exons 4–6 of murine DSG2 (Fig. 1a). This mutation does not disrupt the reading frame and encodes mutant Dsg2 (Dsg2mt) that lacks a substantial segment of the EC1–EC2 domains, which are believed to participate in homo- and heterophilic desmosomal cadherin interactions (Fig. 1b; cf. [28, 59]). Transgenic mice were prepared from embryonal stem cells after homologous recombination of the targeting construct. The neomycin-resistance cassette was then removed with the help of Flpe deleter mice (Fig. 1a) [8]. The resulting DSG2loxP/loxP offspring presented no obvious phenotypic deficiency. To investigate the consequences of constitutive DSG2 mutation, DSG2loxP animals were crossed with transgenic mice producing Cre-recombinase under β-actin promoter control [38]. This led to deletion of exons 4–6 of DSG2 in germ cells and subsequent transmission of the mutated allele to the offspring. In contrast to the previously described DSG2 mutants lacking exons 7 and 8, all of which died around implantation [15], approximately a third of the DSG2mt/mt mice lacking exons 4–6 were born apparently healthy, whereas the rest died during intrauterine development. DSG2mt mRNA and Dsg2mt protein are produced in these mice (Fig. 1c, d). Furthermore, Dsg2mt is located in intercalated discs (Fig. 1e). Dsg2mt expression, however, is considerably lower than that of the wild type (Fig. 1d, e).Fig. 1


Desmoglein 2 mutant mice develop cardiac fibrosis and dilation.

Krusche CA, Holthöfer B, Hofe V, van de Sandt AM, Eshkind L, Bockamp E, Merx MW, Kant S, Windoffer R, Leube RE - Basic Res. Cardiol. (2011)

Preparation of DSG2-mutant mice. a An EcoRV (EV)-segment of the DSG2 wild-type allele (DSG2wt) extending between introns 1 and 8 is depicted at the top. Homologous recombination within this segment leads to the DSG2loxP-neo allele including loxP sites in introns 3 and 6 and an FRT-flanked neomycin-resistance cassette (NFL) in intron 6. Flp-mediated excision of NFL results in allele DSG2loxP. Further recombination by Cre-recombinase generates the mutant allele DSG2mt. Gene regions corresponding to the sequences provided by the targeting construct are indicated by solid lines, and adjoining endogenous gene regions by dotted lines. b Scheme depicting wild-type and mutant Dsg2 (Dsg2wt, Dsg2mt). Dsg2mt lacks parts of the extracellular EC1 and EC2 domains. The scheme further depicts the aminoterminal precursor-specific segment P, the four ~110 amino acid-long extracellular calcium-binding domains EC1–EC4, the extracellular anchor domain EA, transmembrane domain TM, intracellular anchor domain IA, intracellular catenin-binding and cadherin-like sequence ICS, proline-rich linker L, the repeated unit domain RUD and the carboxyterminal tail T. c Detection of mutated DSG2 mRNA. RNA was prepared from DSG2 wild-type and mutant mice (DSG2wt, DSG2mt). GAPDH mRNA was amplified as control. d Analysis of Dsg2 expression by immunoblotting shows a protein band at ~160 kDa in the DSG2wt/wt and of ~110 kDa in DSG2mt/mt mice. The significant difference in mobility is due to deletion of the single glycosylation site. The calculated molecular weight for wild-type Dsg2 is 122,398 and for mutant Dsg2 104,703. Desmocollin 2 (Dsc2), desmoplakin (Dsp) and β-actin served as loading controls. e Immunohistochemistry reveals correct targeting of Dsg2mt to intercalated discs (arrowheads), although expression levels are reduced in DSG2mt/mt mice (same magnification in all images; scale bar, 20 μm). The quantification was done for four wild-type and four mutant 2-week-old littermates from two litters analyzing the ratio of Dsg2 to desmoplakin fluorescence for 40 intercalated discs in each animal
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Fig1: Preparation of DSG2-mutant mice. a An EcoRV (EV)-segment of the DSG2 wild-type allele (DSG2wt) extending between introns 1 and 8 is depicted at the top. Homologous recombination within this segment leads to the DSG2loxP-neo allele including loxP sites in introns 3 and 6 and an FRT-flanked neomycin-resistance cassette (NFL) in intron 6. Flp-mediated excision of NFL results in allele DSG2loxP. Further recombination by Cre-recombinase generates the mutant allele DSG2mt. Gene regions corresponding to the sequences provided by the targeting construct are indicated by solid lines, and adjoining endogenous gene regions by dotted lines. b Scheme depicting wild-type and mutant Dsg2 (Dsg2wt, Dsg2mt). Dsg2mt lacks parts of the extracellular EC1 and EC2 domains. The scheme further depicts the aminoterminal precursor-specific segment P, the four ~110 amino acid-long extracellular calcium-binding domains EC1–EC4, the extracellular anchor domain EA, transmembrane domain TM, intracellular anchor domain IA, intracellular catenin-binding and cadherin-like sequence ICS, proline-rich linker L, the repeated unit domain RUD and the carboxyterminal tail T. c Detection of mutated DSG2 mRNA. RNA was prepared from DSG2 wild-type and mutant mice (DSG2wt, DSG2mt). GAPDH mRNA was amplified as control. d Analysis of Dsg2 expression by immunoblotting shows a protein band at ~160 kDa in the DSG2wt/wt and of ~110 kDa in DSG2mt/mt mice. The significant difference in mobility is due to deletion of the single glycosylation site. The calculated molecular weight for wild-type Dsg2 is 122,398 and for mutant Dsg2 104,703. Desmocollin 2 (Dsc2), desmoplakin (Dsp) and β-actin served as loading controls. e Immunohistochemistry reveals correct targeting of Dsg2mt to intercalated discs (arrowheads), although expression levels are reduced in DSG2mt/mt mice (same magnification in all images; scale bar, 20 μm). The quantification was done for four wild-type and four mutant 2-week-old littermates from two litters analyzing the ratio of Dsg2 to desmoplakin fluorescence for 40 intercalated discs in each animal
Mentions: A targeting construct was prepared for inducible deletion of exons 4–6 of murine DSG2 (Fig. 1a). This mutation does not disrupt the reading frame and encodes mutant Dsg2 (Dsg2mt) that lacks a substantial segment of the EC1–EC2 domains, which are believed to participate in homo- and heterophilic desmosomal cadherin interactions (Fig. 1b; cf. [28, 59]). Transgenic mice were prepared from embryonal stem cells after homologous recombination of the targeting construct. The neomycin-resistance cassette was then removed with the help of Flpe deleter mice (Fig. 1a) [8]. The resulting DSG2loxP/loxP offspring presented no obvious phenotypic deficiency. To investigate the consequences of constitutive DSG2 mutation, DSG2loxP animals were crossed with transgenic mice producing Cre-recombinase under β-actin promoter control [38]. This led to deletion of exons 4–6 of DSG2 in germ cells and subsequent transmission of the mutated allele to the offspring. In contrast to the previously described DSG2 mutants lacking exons 7 and 8, all of which died around implantation [15], approximately a third of the DSG2mt/mt mice lacking exons 4–6 were born apparently healthy, whereas the rest died during intrauterine development. DSG2mt mRNA and Dsg2mt protein are produced in these mice (Fig. 1c, d). Furthermore, Dsg2mt is located in intercalated discs (Fig. 1e). Dsg2mt expression, however, is considerably lower than that of the wild type (Fig. 1d, e).Fig. 1

Bottom Line: Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed.Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue.Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular and Cellular Anatomy, RWTH Aachen University, Wendlingweg 2, Aachen, Germany. ckrusche@ukaachen.de

ABSTRACT
Desmosomes are cell-cell adhesion sites and part of the intercalated discs, which couple adjacent cardiomyocytes. The connection is formed by the extracellular domains of desmosomal cadherins that are also linked to the cytoskeleton on the cytoplasmic side. To examine the contribution of the desmosomal cadherin desmoglein 2 to cardiomyocyte adhesion and cardiac function, mutant mice were prepared lacking a part of the extracellular adhesive domain of desmoglein 2. Most live born mutant mice presented normal overall cardiac morphology at 2 weeks. Some animals, however, displayed extensive fibrotic lesions. Later on, mutants developed ventricular dilation leading to cardiac insufficiency and eventually premature death. Upon histological examination, cardiomyocyte death by calcifying necrosis and replacement by fibrous tissue were observed. Fibrotic lesions were highly proliferative in 2-week-old mutants, whereas the fibrotic lesions of older mutants showed little proliferation indicating the completion of local muscle replacement by scar tissue. Disease progression correlated with increased mRNA expression of c-myc, ANF, BNF, CTGF and GDF15, which are markers for cardiac stress, remodeling and heart failure. Taken together, the desmoglein 2-mutant mice display features of dilative cardiomyopathy and arrhythmogenic right ventricular cardiomyopathy, an inherited human heart disease with pronounced fibrosis and ventricular arrhythmias that has been linked to mutations in desmosomal proteins including desmoglein 2.

Show MeSH
Related in: MedlinePlus