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Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W - J. Cell Biol. (2004)

Bottom Line: Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrueck Center for Molecular Medicine (MDC), D-13092 Berlin, Germany.

ABSTRACT
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

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Major architectural and compositional changes of the adhering junctions in the intercalated disks of hearts in plakophilin 2–deficient E10.75 mouse embryos. Laser-scanning, double-label immunofluorescence microscopy of cryostat sections through cardiac tissue, performed with wt and plakophilin 2–deficient (pkp2−/−) embryos. Specific combinations of antibodies were used: rabbit antibodies to desmoplakin (DP, marked by green fluorescence) and murine mAbs to diverse other cardiac adhering junction components (red fluorescence): (a and a′) plakoglobin (PG); (b and b′) N-cadherin (N-Cad); (c and c′) β-catenin (β-Cat); (d and d′) plakophilin 2 (PKP2); (e and e′) desmoglein 2 (Dsg2). The merged fluorescence images are shown in the right-hand column. (A) In the wt embryos, the special adhering junctions of the intercalated disks show far-reaching colocalization (yellow) of desmoplakin with (a) plakoglobin, (b) N-cadherin, (c) β-catenin, (d) plakophilin 2, and (e) desmoglein 2. Other known plaque proteins of cardiac adhering junctions show identical localization, including p120ctn and α-catenin (see also Janssens et al., 2001). (B) By contrast, in the pkp2−/− embryos none of the plaque proteins colocalize with desmoplakin (a′–e′). In the mutants, desmoplakin is dispersed over the cytoplasm, often appearing in granular aggregates (green dots). (d′) Complete absence of plakophilin 2 immunostaining in the mutant embryos is shown. (e′) Note that Dsg2 is not detectably enriched in the intercalated disks of the mutants. Bars, 50 μm.
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fig4: Major architectural and compositional changes of the adhering junctions in the intercalated disks of hearts in plakophilin 2–deficient E10.75 mouse embryos. Laser-scanning, double-label immunofluorescence microscopy of cryostat sections through cardiac tissue, performed with wt and plakophilin 2–deficient (pkp2−/−) embryos. Specific combinations of antibodies were used: rabbit antibodies to desmoplakin (DP, marked by green fluorescence) and murine mAbs to diverse other cardiac adhering junction components (red fluorescence): (a and a′) plakoglobin (PG); (b and b′) N-cadherin (N-Cad); (c and c′) β-catenin (β-Cat); (d and d′) plakophilin 2 (PKP2); (e and e′) desmoglein 2 (Dsg2). The merged fluorescence images are shown in the right-hand column. (A) In the wt embryos, the special adhering junctions of the intercalated disks show far-reaching colocalization (yellow) of desmoplakin with (a) plakoglobin, (b) N-cadherin, (c) β-catenin, (d) plakophilin 2, and (e) desmoglein 2. Other known plaque proteins of cardiac adhering junctions show identical localization, including p120ctn and α-catenin (see also Janssens et al., 2001). (B) By contrast, in the pkp2−/− embryos none of the plaque proteins colocalize with desmoplakin (a′–e′). In the mutants, desmoplakin is dispersed over the cytoplasm, often appearing in granular aggregates (green dots). (d′) Complete absence of plakophilin 2 immunostaining in the mutant embryos is shown. (e′) Note that Dsg2 is not detectably enriched in the intercalated disks of the mutants. Bars, 50 μm.

Mentions: We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).


Requirement of plakophilin 2 for heart morphogenesis and cardiac junction formation.

Grossmann KS, Grund C, Huelsken J, Behrend M, Erdmann B, Franke WW, Birchmeier W - J. Cell Biol. (2004)

Major architectural and compositional changes of the adhering junctions in the intercalated disks of hearts in plakophilin 2–deficient E10.75 mouse embryos. Laser-scanning, double-label immunofluorescence microscopy of cryostat sections through cardiac tissue, performed with wt and plakophilin 2–deficient (pkp2−/−) embryos. Specific combinations of antibodies were used: rabbit antibodies to desmoplakin (DP, marked by green fluorescence) and murine mAbs to diverse other cardiac adhering junction components (red fluorescence): (a and a′) plakoglobin (PG); (b and b′) N-cadherin (N-Cad); (c and c′) β-catenin (β-Cat); (d and d′) plakophilin 2 (PKP2); (e and e′) desmoglein 2 (Dsg2). The merged fluorescence images are shown in the right-hand column. (A) In the wt embryos, the special adhering junctions of the intercalated disks show far-reaching colocalization (yellow) of desmoplakin with (a) plakoglobin, (b) N-cadherin, (c) β-catenin, (d) plakophilin 2, and (e) desmoglein 2. Other known plaque proteins of cardiac adhering junctions show identical localization, including p120ctn and α-catenin (see also Janssens et al., 2001). (B) By contrast, in the pkp2−/− embryos none of the plaque proteins colocalize with desmoplakin (a′–e′). In the mutants, desmoplakin is dispersed over the cytoplasm, often appearing in granular aggregates (green dots). (d′) Complete absence of plakophilin 2 immunostaining in the mutant embryos is shown. (e′) Note that Dsg2 is not detectably enriched in the intercalated disks of the mutants. Bars, 50 μm.
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fig4: Major architectural and compositional changes of the adhering junctions in the intercalated disks of hearts in plakophilin 2–deficient E10.75 mouse embryos. Laser-scanning, double-label immunofluorescence microscopy of cryostat sections through cardiac tissue, performed with wt and plakophilin 2–deficient (pkp2−/−) embryos. Specific combinations of antibodies were used: rabbit antibodies to desmoplakin (DP, marked by green fluorescence) and murine mAbs to diverse other cardiac adhering junction components (red fluorescence): (a and a′) plakoglobin (PG); (b and b′) N-cadherin (N-Cad); (c and c′) β-catenin (β-Cat); (d and d′) plakophilin 2 (PKP2); (e and e′) desmoglein 2 (Dsg2). The merged fluorescence images are shown in the right-hand column. (A) In the wt embryos, the special adhering junctions of the intercalated disks show far-reaching colocalization (yellow) of desmoplakin with (a) plakoglobin, (b) N-cadherin, (c) β-catenin, (d) plakophilin 2, and (e) desmoglein 2. Other known plaque proteins of cardiac adhering junctions show identical localization, including p120ctn and α-catenin (see also Janssens et al., 2001). (B) By contrast, in the pkp2−/− embryos none of the plaque proteins colocalize with desmoplakin (a′–e′). In the mutants, desmoplakin is dispersed over the cytoplasm, often appearing in granular aggregates (green dots). (d′) Complete absence of plakophilin 2 immunostaining in the mutant embryos is shown. (e′) Note that Dsg2 is not detectably enriched in the intercalated disks of the mutants. Bars, 50 μm.
Mentions: We generated a mutation of the plakophilin 2 gene by homologous recombination in embryonic stem (ES) cells. In the targeting vector, a neo cassette inserted in opposite transcriptional orientation replaced a 7.8-kb genomic fragment from the NotI site in exon 1 to the BamHI site in intron 1 (Fig. 1 a). Homologous recombination events were identified by Southern blot analyses (Fig. 1 b): an external probe (ext) yielded a novel Xba fragment of 10 kb in the mutant, Δpkp2 (the wild-type [wt] fragment is 16 kb), and the neo probe produced a 4.5-kb HindIII fragment. We generated a mutation in the plakophilin 2 gene after homologous recombination because insertion of neo prevents splicing between exon 1 and 2 and leads to an early stop of plakophilin 2 translation after 43 aa. Using two lines of mutant ES cells, we produced plakophilin 2 mutant chimeric and heterozygous mice that were healthy and fertile. However, matings between heterozygous mice produced no live plakophilin 2–deficient offspring, implying that the mutant embryos died during embryogenesis. To determine the time of death, embryos from different developmental stages were genotyped by PCR (Fig. 1 c) and inspected visually. Up to day 10.75 of embryogenesis (E10.75), the expected Mendelian ratio of homozygous mutant embryos was observed (Table I); however the mutant embryos showed blood accumulation in the pericardial and peritoneal cavities. At E11.5, the number of viable plakophilin 2 −/− embryos declined, as judged by PCR genotyping and lack of heart beating. Western blot analyses using an antibody against the COOH terminus of plakophilin 2 indicated absence of full-length or truncated protein in E10.75 plakophilin 2 −/− embryos (Fig. 1 d). In wt embryos at E10.75, plakophilin 2 was prominent in cardiomyocytes of the atrium and the ventricle of the heart (Fig. 1 e), and expression was lost in the mutation embryos (see also below, Fig. 4 Bd′, and Fig. 8). At E13.75, plakophilin 2 was intensely synthesized in the wt cardiomyocytes, but less in the surrounding epicard (Fig. 1 f).

Bottom Line: Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease.By contrast, embryonic epithelia show normal junctions.Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

View Article: PubMed Central - PubMed

Affiliation: Max Delbrueck Center for Molecular Medicine (MDC), D-13092 Berlin, Germany.

ABSTRACT
Plakophilins are proteins of the armadillo family that function in embryonic development and in the adult, and when mutated can cause disease. We have ablated the plakophilin 2 gene in mice. The resulting mutant mice exhibit lethal alterations in heart morphogenesis and stability at mid-gestation (E10.5-E11), characterized by reduced trabeculation, disarrayed cytoskeleton, ruptures of cardiac walls, and blood leakage into the pericardiac cavity. In the absence of plakophilin 2, the cytoskeletal linker protein desmoplakin dissociates from the plaques of the adhering junctions that connect the cardiomyocytes and forms granular aggregates in the cytoplasm. By contrast, embryonic epithelia show normal junctions. Thus, we conclude that plakophilin 2 is important for the assembly of junctional proteins and represents an essential morphogenic factor and architectural component of the heart.

Show MeSH
Related in: MedlinePlus