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Werner W. Franke - One of the best experts on this subject based on the ideXlab platform.
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the area composita of adhering junctions connecting heart muscle cells of vertebrates v the importance of Plakophilin 2 demonstrated by small interference rna mediated knockdown in cultured rat cardiomyocytes
European Journal of Cell Biology, 2008Co-Authors: Sebastian Pieperhoff, Heiderose Schumacher, Werner W. FrankeAbstract:In the adult mammalian heart, the cardiomyocytes are connected by large polar arrays of closely spaced or even fused composite, plaque-bearing adhering junctions (areae compositae, ACs), in a region usually termed “intercalated disk” (ID). We have recently reported that during late embryogenesis and postnatally these polar assemblies of AC-junction structures are gradually formed as replacements of distinct embryonal junctions representing desmosomes and fasciae adhaerentes which then may amalgamate to the fused AC structures, in some regions occupying more than 90% of the total ID area. Previous gene knockout results as well as mutation analyses of specific human cardiomyopathies have suggested that among the various AC constituents, the desmosomal plaque protein, Plakophilin-2, plays a particularly important role in the formation, architectural organization and stability of these junctions interconnecting mature cardiomyocytes. To examine this hypothesis, we have decided to study losses of – or molecular alterations in – such AC proteins with respect to their effects on myocardiac organization and functions. Here we report that Plakophilin-2 is indeed of obvious importance for myocardial architecture and cell–cell coupling of rat cardiomyocytes growing in culture. We show that siRNA-mediated reduction of the cardiomyocyte content of Plakophilin-2 but not of some other major plaque components such as desmoplakin results in progressive disintegration – and losses – of AC junction structures and that numerous variously sized vesicles appear, which are plaque protein-associated as demonstrable by immunofluorescence and immunoelectron microscopy. The importance of Plakophilin-2 as a kind of “organizer” protein in the formation, stabilization and functions of the AC structure and the ID architecture is discussed in relation to other junction proteins and to causes of certain cardiomyopathies.
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Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules
Molecular biology of the cell, 2006Co-Authors: Ilse Hofmann, Martina Schnolzer, Marialuisa Casella, Tanja Schlechter, Herbert Spring, Werner W. FrankeAbstract:Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.
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requirement of Plakophilin 2 for heart morphogenesis and cardiac junction formation
Journal of Cell Biology, 2004Co-Authors: Katja S Grossmann, Werner W. Franke, Christine Grund, Joerg Huelsken, Martin Behrend, Bettina Erdmann, Walter BirchmeierAbstract: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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De novo formation of desmosomes in cultured cells upon transfection of genes encoding specific desmosomal components.
Experimental Cell Research, 2003Co-Authors: Joachim Koeser, Sergey M. Troyanovsky, Christine Grund, Werner W. FrankeAbstract:Abstract Desmosomes are cell junctions and cytoskeleton-anchoring structures of epithelia, the myocardium, and dendritic reticulum cells of lymphatic follicles whose major components are known. Using cultured HT-1080 SL-1 fibrosarcoma-derived cells and transfection of cDNAs encoding specific desmosomal components, we have determined a minimum ensemble of proteins sufficient to introduce de novo structures, which, by morphology and functional competence, are indistinguishable from authentic desmosomes. In a more refined analysis, the influence of the desmosomal proteins desmoplakin (Dp), plakoglobin (Pg), and Plakophilin 2 (Pp2) on the lateral clustering of the desmosomal transmembrane-glycoprotein desmoglein 2 (Dsg) was examined. We found that for efficient clustering of desmoglein 2 and desmosome structure formation, all three major plaque proteins—desmoplakin, plakoglobin, and Plakophilin 2— were necessary. Furthermore, in this cell model, Plakophilin 2 was capable of directing desmoplakin to adhaerens junctions (AJ), whereas plakoglobin was crucial for the segregation of desmosomal and AJ components. These results are discussed with respect to the variability in cell junction composition observed in various nonepithelial tissues.
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nuclear particles containing rna polymerase iii complexes associated with the junctional plaque protein Plakophilin 2
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Claudia Mertens, Ilse Hofmann, Martina Schnolzer, Zhengxin Wang, Martin Teichmann, Setareh Sepehri Chong, Werner W. FrankeAbstract:Plakophilin 2, a member of the arm-repeat protein family, is a dual location protein that occurs both in the cytoplasmic plaques of desmosomes as an architectural component and in an extractable form in the nucleoplasm. Here we report the existence of two nuclear particles containing Plakophilin 2 and the largest subunit of RNA polymerase (pol) III (RPC155), both of which colocalize and are coimmunoselected with other pol III subunits and with the transcription factor TFIIIB. We also show that Plakophilin 2 is present in the pol III holoenzyme, but not the core complex, and that it binds specifically to RPC155 in vitro. We propose the existence of diverse nuclear particles in which proteins known as plaque proteins of intercellular junctions are complexed with specific nuclear proteins.
Mario Delmar - One of the best experts on this subject based on the ideXlab platform.
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In silico Identification of Disrupted Myocardial Calcium Homeostasis as Proarrhythmic Trigger in Arrhythmogenic Cardiomyopathy
'Frontiers Media SA', 2021Co-Authors: Aurore Lyon, Mario Delmar, Chantal J. M. Van Opbergen, Jordi Heijman, Toon A. B. Van VeenAbstract:Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in Plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to Plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear.Aim: To unravel the mechanisms by which calcium-handling abnormalities in Plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy.Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from Plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model.Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings.Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of Plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to Plakophilin-2 loss-of-function
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Cellular Biology Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes
2016Co-Authors: Priscila Y Sato, Lori L Isom, Steven M Taffet, Hassan Musa, A Coombs, Guadalupe Guerrero-serna, Gustavo A. Patiño, Mario DelmarAbstract:Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed. (Circ Res. 2009;105:523-526.) Key Words: Plakophilin-2 intercalated disc arrhythmogenic right ventricular cardiomyopathy cardiac desmosomes A high-resolution image of the site of end–end contactbetween cardiomyocytes reveals an electron-dense or-ganization called “the intercalated disc. ” Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the inter-calated disc. Among them is NaV1.5, the major subunit of the cardiac sodium channel.1 Here, we ask whether Nav1.5 and the desmosomal protein Plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a func-tional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte
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interactions between ankyrin g Plakophilin 2 and connexin43 at the cardiac intercalated disc
Circulation Research, 2011Co-Authors: Priscila Y Sato, Wanda Coombs, Oxana Nekrasova, Lori L Isom, Steven M Taffet, Mario DelmarAbstract:Rationale:The early description of the intercalated disc defined 3 structures, all of them involved in cell-cell communication: desmosomes, gap junctions, and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells also populate the intercalated disc. Key among them is the voltage-gated sodium channel complex. An important component of this complex is the cytoskeletal adaptor protein Ankyrin-G (AnkG). Objective:To test the hypothesis that AnkG partners with desmosome and gap junction molecules and exerts a functional effect on intercellular communication in the heart. Methods and Results:We used a combination of microscopy, immunochemistry, patch-clamp, and optical mapping to assess the interactions between AnkG, Plakophilin-2, and Connexin43. Coimmunoprecipitation studies from rat heart lysate demonstrated associations between the 3 molecules. With the use of siRNA technology, we demonstrated that loss of AnkG expression caused signifi...
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loss of Plakophilin 2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes
Circulation Research, 2009Co-Authors: Priscila Y Sato, Guadalupe Guerreroserna, Gustavo A Patino, Wanda Coombs, Lori L Isom, Steven M Taffet, Hassan Musa, Mario DelmarAbstract:Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.
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molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia cardiomyopathy
Heart Rhythm, 2007Co-Authors: Eva M Oxford, Jeffrey E. Saffitz, Wanda Coombs, Steven M Taffet, Melanie D Everitt, Sydney N Moise, Anna R M Gelzer, Marc S Kraus, Mario DelmarAbstract:Background Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. Objective We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). Methods We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, Plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). Results In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or Plakophilin 2. Conclusion Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell–cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.
R A J Eady - One of the best experts on this subject based on the ideXlab platform.
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genotype phenotype correlation in skin fragility ectodermal dysplasia syndrome resulting from mutations in Plakophilin 1
Experimental Dermatology, 2002Co-Authors: T Hamada, Andrew P South, Yoshihiko Mitsuhashi, T Kinebuchi, O Bleck, G H S Ashton, Y Hozumi, Tamio Suzuki, Takashi Hashimoto, R A J EadyAbstract:We report a 42-year-old Japanese man with an unusual autosomal recessive genodermatosis. The clinical features comprised normal skin at birth, loss of scalp hair at 3-months of age after a febrile illness, progressive nail dystrophy during infancy, palmoplantar keratoderma starting around the age of 18 years and trauma-induced skin fragility and blisters noted from the age of 20 years. Skin biopsy of rubbed non-lesional skin revealed widening of spaces between adjacent keratinocytes from the suprabasal layer upwards. Electron microscopy demonstrated a reduced number of hypoplastic desmosomes. Immunohistochemical labeling showed a reduction in intercellular staining for the desmosome component Plakophilin 1. Mutation analysis revealed a homozygous intron 11 donor splice site mutation in the Plakophilin 1 gene, 2021+1 G>A (GenBank no. Z34974). RT-PCR, using RNA extracted from the skin biopsy, provided evidence for residual low levels of the full-length wild-type transcript (approximately 8%) as well as multiple other near full-length transcripts, one of which was in frame leading to deletion of 17 amino acids from the 9th arm-repeat unit of the Plakophilin 1 tail domain. Thus, the molecular findings help explain the clinical features in the patient, who has a similar but milder phenotype to previously reported patients with skin fragility-ectodermal dysplasia syndrome associated with complete ablation of Plakophilin 1 (OMIM 604536). This new 'mitis' phenotype provides further clinicopathological evidence for the role of Plakophilin 1 in keratinocyte cell-cell adhesion and ectodermal development.
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preimplantation genetic diagnosis of compound heterozygous mutations leading to ablation of Plakophilin 1 pkp1 and resulting in skin fragility ectodermal dysplasia syndrome a case report
Prenatal Diagnosis, 2000Co-Authors: Alan R Thornhill, Susan J. Pickering, R A J Eady, Neil V Whittock, Jenny Caller, Vicky Andritsos, H Bickerstaff, Alan H Handyside, Peter BraudeAbstract:A new form of genodermatosis resulting from mutations in the gene Plakophilin 1 (PKP1) has recently been identified. The clinical features of a functional knockout of PKP1 are a combination of skin fragility and a form of hypohydrotic ectodermal dysplasia. We have developed a single cell polymerase chain reaction (PCR) assay suitable for preimplantation genetic diagnosis (PGD) and here we report on the clinical application of this assay.
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genomic amplification of the human Plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia skin fragility syndrome
Journal of Investigative Dermatology, 2000Co-Authors: Neil V Whittock, Marek Haftek, Nathalie Angoulvant, F Wolf, H Perrot, R A J EadyAbstract:Summary Ectodermal dysplasia/skin fragility syndrome is a recently described autosomal recessive disease affecting skin, nails, and hair (MIM 604536), that results from mutations in Plakophilin 1 , a structural component of desmosomes. We report a new Plakophilin 1 mutation in an affected patient as well as detailing the intron–exon organization of the gene to facilitate future polymerase chain reaction-based mutation screening. Using polymerase chain reaction amplification of genomic DNA, we identified 15 exons spanning approximately 50 kb. Direct sequencing disclosed several nonpathogenic intragenic polymorphisms, as well as a homozygous splice site mutation (1233–2 A→T; GenBank Z73678) in a 17 y old affected male. The clinical features comprised skin erosions, dystrophic nails, sparse hair, and painful thickening and cracking of palms and soles. Skin biopsy showed negative immunolabeling with an anti- Plakophilin 1 antibody and small desmosomes. These results expand the database of Plakophilin 1 mutations and demonstrate the importance of this protein in the stabilization of desmosomal adhesion in terminally differentiating keratinocytes.
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mutations in the Plakophilin 1 gene result in ectodermal dysplasia skin fragility syndrome
Nature Genetics, 1997Co-Authors: John A Mcgrath, James R Mcmillan, I M Leigh, Carrie S Shemanko, S K Runswick, E B Lane, David R Garrod, R A J EadyAbstract:Members of the armadillo protein gene family, which includes plakoglobin and beta-catenin, have important functions in cytoskeleton/cell membrane interactions. These proteins may act as linker molecules at adherens junctions and desmosomes at the plasma membrane; in addition, they may have pivotal roles in signal transduction pathways and significant effects on cell behaviour during development. Here, we describe the first human mutations in one of these dual function proteins, Plakophilin 1 (band-6 protein; refs 8-10). The affected individual has a complete absence of immunostaining for Plakophilin 1 in the skin and is a compound heterozygote for autosomal-recessively inherited premature termination codons of translation on both alleles of the Plakophilin 1 gene (PKP1). Clinically, there are features of both cutaneous fragility and congenital ectodermal dysplasia affecting skin, hair and nails. There is no evidence of significant abnormalities in other epithelia or tissues. Desmosomes in the skin are small and poorly formed with widening of keratinocyte intercellular spaces and perturbed desmosome/keratin intermediate filament interactions. The molecular findings and clinical observations in this patient attest to the dual importance of Plakophilin 1 in both cutaneous cell-call adhesion and epidermal morphogenesis.
Neil V Whittock - One of the best experts on this subject based on the ideXlab platform.
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preimplantation genetic diagnosis of skin fragility ectodermal dysplasia syndrome
British Journal of Dermatology, 2006Co-Authors: Hiva Fassihi, Jan Grace, Alison Lashwood, Neil V WhittockAbstract:Skin fragility-ectodermal dysplasia syndrome is an autosomal recessive disorder caused by loss-of-function mutations in the desmosomal protein, Plakophilin 1. Clinically, there may be considerable morbidity from extensive skin erosions and painful fissures on the palms and soles. In the absence of any specific treatment, prenatal diagnosis is an option for couples at reproductive risk of recurrence. In 2000, we developed and applied a single cell nested polymerase chain reaction protocol to test one couple for compound heterozygous Plakophilin 1 gene mutations by preimplantation genetic diagnosis (PGD). Although pregnancy was established, an unrelated trisomy 22 led to a spontaneous abortion. However, eight embryos of known genetic status were cryopreserved at that stage, and we planned to undertake subsequent frozen embryo replacement cycles that might lead to the birth of an unaffected child in this family. Embryo cryopreservation was carried out in June 2000 using standard protocols in a three-step freezing procedure. Four embryos were thawed in March 2003, one of which was viable and was used in a frozen embryo replacement cycle, but pregnancy did not occur. The remaining four embryos were thawed in February 2004, two of which were viable (both carriers of the paternal mutation) and these were used in a second frozen embryo replacement cycle, and a singleton pregnancy was established. The child's Plakophilin 1 genotype was assessed by direct nucleotide sequencing across the site of both potential mutations. Following two frozen embryo replacement cycles, and almost 4 years after the initial embryo biopsy and mutation analysis, a pregnancy was achieved that progressed to term with the birth of a healthy baby girl. Nucleotide sequencing of cord blood DNA, taken immediately after delivery, showed that the child was a heterozygous carrier of the paternal mutation but not of the maternal mutation. This case demonstrates the value of embryo cryopreservation, which can increase the number of embryo replacement procedures and hence the cumulative pregnancy rate per retrieval cycle. Moreover, this is the first report of successful full-term pregnancy and birth of a healthy baby following exclusion of a severe genodermatosis by PGD. The successful outcome of PGD in this case illustrates what is technically possible for couples at risk of recurrence of a severe inherited skin disease.
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preimplantation genetic diagnosis of compound heterozygous mutations leading to ablation of Plakophilin 1 pkp1 and resulting in skin fragility ectodermal dysplasia syndrome a case report
Prenatal Diagnosis, 2000Co-Authors: Alan R Thornhill, Susan J. Pickering, R A J Eady, Neil V Whittock, Jenny Caller, Vicky Andritsos, H Bickerstaff, Alan H Handyside, Peter BraudeAbstract:A new form of genodermatosis resulting from mutations in the gene Plakophilin 1 (PKP1) has recently been identified. The clinical features of a functional knockout of PKP1 are a combination of skin fragility and a form of hypohydrotic ectodermal dysplasia. We have developed a single cell polymerase chain reaction (PCR) assay suitable for preimplantation genetic diagnosis (PGD) and here we report on the clinical application of this assay.
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genomic amplification of the human Plakophilin 1 gene and detection of a new mutation in ectodermal dysplasia skin fragility syndrome
Journal of Investigative Dermatology, 2000Co-Authors: Neil V Whittock, Marek Haftek, Nathalie Angoulvant, F Wolf, H Perrot, R A J EadyAbstract:Summary Ectodermal dysplasia/skin fragility syndrome is a recently described autosomal recessive disease affecting skin, nails, and hair (MIM 604536), that results from mutations in Plakophilin 1 , a structural component of desmosomes. We report a new Plakophilin 1 mutation in an affected patient as well as detailing the intron–exon organization of the gene to facilitate future polymerase chain reaction-based mutation screening. Using polymerase chain reaction amplification of genomic DNA, we identified 15 exons spanning approximately 50 kb. Direct sequencing disclosed several nonpathogenic intragenic polymorphisms, as well as a homozygous splice site mutation (1233–2 A→T; GenBank Z73678) in a 17 y old affected male. The clinical features comprised skin erosions, dystrophic nails, sparse hair, and painful thickening and cracking of palms and soles. Skin biopsy showed negative immunolabeling with an anti- Plakophilin 1 antibody and small desmosomes. These results expand the database of Plakophilin 1 mutations and demonstrate the importance of this protein in the stabilization of desmosomal adhesion in terminally differentiating keratinocytes.
Steven M Taffet - One of the best experts on this subject based on the ideXlab platform.
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Cellular Biology Loss of Plakophilin-2 Expression Leads to Decreased Sodium Current and Slower Conduction Velocity in Cultured Cardiac Myocytes
2016Co-Authors: Priscila Y Sato, Lori L Isom, Steven M Taffet, Hassan Musa, A Coombs, Guadalupe Guerrero-serna, Gustavo A. Patiño, Mario DelmarAbstract:Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed. (Circ Res. 2009;105:523-526.) Key Words: Plakophilin-2 intercalated disc arrhythmogenic right ventricular cardiomyopathy cardiac desmosomes A high-resolution image of the site of end–end contactbetween cardiomyocytes reveals an electron-dense or-ganization called “the intercalated disc. ” Its classic definition involves 3 structures: desmosomes and adherens junctions, providing mechanical coupling; and gap junctions, allowing electric/metabolic synchronization between cells. Recent studies show that other molecules, not directly involved in intercellular coupling, also reside preferentially at the inter-calated disc. Among them is NaV1.5, the major subunit of the cardiac sodium channel.1 Here, we ask whether Nav1.5 and the desmosomal protein Plakophilin-2 (PKP2) coexist in the same molecular complex and whether loss of PKP2 expression affects (1) the amplitude and kinetics of the sodium current and (2) action potential propagation in a monolayer of cardiomyocytes. Our data demonstrate a func-tional crosstalk between a protein defined in the context of intercellular junctions (PKP2) and another protein that is fundamental to the electrical behavior of the single myocyte
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interactions between ankyrin g Plakophilin 2 and connexin43 at the cardiac intercalated disc
Circulation Research, 2011Co-Authors: Priscila Y Sato, Wanda Coombs, Oxana Nekrasova, Lori L Isom, Steven M Taffet, Mario DelmarAbstract:Rationale:The early description of the intercalated disc defined 3 structures, all of them involved in cell-cell communication: desmosomes, gap junctions, and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells also populate the intercalated disc. Key among them is the voltage-gated sodium channel complex. An important component of this complex is the cytoskeletal adaptor protein Ankyrin-G (AnkG). Objective:To test the hypothesis that AnkG partners with desmosome and gap junction molecules and exerts a functional effect on intercellular communication in the heart. Methods and Results:We used a combination of microscopy, immunochemistry, patch-clamp, and optical mapping to assess the interactions between AnkG, Plakophilin-2, and Connexin43. Coimmunoprecipitation studies from rat heart lysate demonstrated associations between the 3 molecules. With the use of siRNA technology, we demonstrated that loss of AnkG expression caused signifi...
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loss of Plakophilin 2 expression leads to decreased sodium current and slower conduction velocity in cultured cardiac myocytes
Circulation Research, 2009Co-Authors: Priscila Y Sato, Guadalupe Guerreroserna, Gustavo A Patino, Wanda Coombs, Lori L Isom, Steven M Taffet, Hassan Musa, Mario DelmarAbstract:Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (NaV1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with NaV1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.
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molecular composition of the intercalated disc in a spontaneous canine animal model of arrhythmogenic right ventricular dysplasia cardiomyopathy
Heart Rhythm, 2007Co-Authors: Eva M Oxford, Jeffrey E. Saffitz, Wanda Coombs, Steven M Taffet, Melanie D Everitt, Sydney N Moise, Anna R M Gelzer, Marc S Kraus, Mario DelmarAbstract:Background Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by ventricular arrhythmias, sudden death, and fatty or fibrofatty replacement of right ventricular myocytes. Recent studies have noted an association between human ARVD/C and molecular remodeling of intercalated disc structures. However, progress has been constrained by limitations inherent to human studies. Objective We studied the molecular composition of the intercalated disc structure in a naturally occurring animal model of ARVD/C (Boxer dogs). Methods We studied hearts from 12 Boxers with confirmed ARVD/C and 2 controls. Ventricular sections from 4 animals were examined by immunofluorescent microscopy. Frozen tissue samples were used for Western blot analysis. Proteins investigated were N-cadherin, Plakophilin 2, desmoplakin, plakoglobin, desmin, and connexin 43 (Cx43). Results In control dogs, all proteins tested by immunofluorescence analysis yielded intense localized signals at sites of end-to-end cell apposition. In contrast, myocardial tissues from ARVD/C-afflicted Boxers showed preservation of N-cadherin staining but loss of detectable signal for Cx43 at the intercalated disc location. Western blots indicated that the Cx43 protein was still present in the samples. Gene sequencing analysis showed no mutations in desmoplakin, plakoglobin, Cx43, or Plakophilin 2. Conclusion Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell–cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.