Decreased Expression of Plakophilin-2 and αT-Catenin in Arrhythmogenic Right Ventricular Cardiomyopathy: Potential Markers for Diagnosis.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary disease of the heart muscle. Clinical challenges remain, however, in identifying patients with ARVC in the early or concealed stages with subtle clinical manifestations. Therefore, we wanted to identify potential targets by immunohistochemical (IHC) analysis in comparison with controls. Pathogenic mutations were identified in 11 of 37 autopsied patients with ARVC. As observed from IHC analysis of the RV, expression of αT-catenin and plakophilin-2 is significantly decreased in autopsied patients with ARVC as compared to controls, and the decreased expression is consistent in patients with and without pathogenic mutations. Furthermore, ARVC specimens demonstrated a reduced localization of αT-catenin, desmocollin-2, desmoglein-2, desmoplakin, and plakophilin-2 on intercalated discs. These findings have been validated by comparing RV specimens obtained via endomyocardial biopsy between patients with ARVC and those without. The pathogenic mutation was present in 3 of 5 clinical patients with ARVC. In HL-1 myocytes, siRNA was used to knockdown CTNNA3, and western blotting analysis demonstrated that the decline in αT-catenin expression was accompanied by a significant decline in the expression of plakophilin-2. The aforementioned effect was directed towards protein degradation rather than mRNA stability. Plakophilin-2 expression decreases concurrently with the decline in CTNNA3 expression. Therefore, the expression of αT-catenin and plakophilin-2 could be potential surrogates for the diagnosis of ARVC.

Plakophilin 1-deficient cells upregulate SPOCK1: implications for prostate cancer progression.

Plakophilin (PKP) 1 is frequently downregulated in prostate cancer and therefore may play a tumor-suppressive role. In the present study, we stably knocked down PKP1 in the non-neoplastic, prostatic BPH-1 cell line. In the PKP1-deficient cells, the expression of keratin 14 was lost, and the apoptosis rate was significantly reduced indicating that the cells acquired new biological capabilities. Moreover, we analyzed the gene expression profile of the PKP1-deficient BPH-1 cells. Among the genes that were significantly altered upon PKP1 knockdown, we noticed several extracellular matrix (ECM)-related genes and identified sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1/testican-1) as a gene of interest. SPOCK1 is a component of the ECM and belongs to a matricellular protein family named secreted protein, acidic, cysteine-rich (SPARC). The role of SPOCK1 in prostate cancer has not been clearly elucidated. We analyzed SPOCK1 mRNA expression levels in different cancer databases and characterized its expression in 136 prostatic adenocarcinomas by immunohistochemistry and western blot. SPOCK1 revealed a cytoplasmic localization in the glandular epithelium of the prostate and showed a significant upregulation of mRNA and protein in prostate tumor samples. Our findings support the hypothesis that PKP1 may have a tumor-suppressive function and suggest an important role of SPOCK1 in prostate tumor progression. Collectively, altered expression of PKP1 and SPOCK1 appears to be a frequent and critical event in prostate cancer.

Exercise triggers ARVC phenotype in mice expressing a disease-causing mutated version of human plakophilin-2.

BACKGROUND: Exercise has been proposed as a trigger for arrhythmogenic right ventricular cardiomyopathy (ARVC) phenotype manifestation; however, research is hampered by the limited availability of animal models in which disease-associated mutations can be tested. OBJECTIVES: This study evaluated the impact of exercise on ARVC cardiac manifestations in mice after adeno-associated virus (AAV)-mediated gene delivery of mutant human PKP2, which encodes the desmosomal protein plakophilin-2. METHODS: We developed a new model of cardiac tissue-specific transgenic-like mice on the basis of AAV gene transfer to test the potential of a combination of a human PKP2 mutation and endurance training to trigger an ARVC-like phenotype. RESULTS: Stable cardiac expression of mutant PKP2 (c.2203C>T), encoding the R735X mutant protein, was achieved 4 weeks after a single AAV9-R735X intravenous injection. High-field cardiac magnetic resonance over a 10-month postinfection follow-up did not detect an overt right ventricular (RV) phenotype in nonexercised (sedentary) mice. In contrast, endurance exercise training (initiated 2 weeks after AAV9-R735X injection) resulted in clear RV dysfunction that resembled the ARVC phenotype (impaired global RV systolic function and RV regional wall motion abnormalities on cardiac magnetic resonance). At the histological level, RV samples from endurance-trained R735X-infected mice displayed connexin 43 delocalization at intercardiomyocyte gap junctions, a change not observed in sedentary mice. CONCLUSIONS: The introduction of the PKP2 R735X mutation into mice resulted in an exercise-dependent ARVC phenotype. The R735X mutation appears to function as a dominant-negative variant. This novel system for AAV-mediated introduction of a mutation into wild-type mice has broad potential for study of the implication of diverse mutations in complex cardiomyopathies.

Deficient plakophilin-1 expression due to a mutation in PKP1 causes ectodermal dysplasia-skin fragility syndrome in Chesapeake Bay retriever dogs.

In humans, congenital and hereditary skin diseases associated with epidermal cell-cell separation (acantholysis) are very rare, and spontaneous animal models of these diseases are exceptional. Our objectives are to report a novel congenital acantholytic dermatosis that developed in Chesapeake Bay retriever dogs. Nine affected puppies in four different litters were born to eight closely related clinically normal dogs. The disease transmission was consistent with an autosomal recessive mode of inheritance. Clinical signs occurred immediately after birth with superficial epidermal layers sloughing upon pressure. At three month of age, dogs exhibited recurrent superficial skin sloughing and erosions at areas of friction and mucocutaneous junctions; their coat was also finer than normal and there were patches of partial hair loss. At birth, histopathology revealed severe suprabasal acantholysis, which became less severe with ageing. Electron microscopy demonstrated a reduced number of partially formed desmosomes with detached and aggregated keratin intermediate filaments. Immunostaining for desmosomal adhesion molecules revealed a complete lack of staining for plakophilin-1 and anomalies in the distribution of desmoplakin and keratins 10 and 14. Sequencing revealed a homozygous splice donor site mutation within the first intron of PKP1 resulting in a premature stop codon, thereby explaining the inability to detect plakophilin-1 in the skin. Altogether, the clinical and pathological findings, along with the PKP1 mutation, were consistent with the diagnosis of ectodermal dysplasia-skin fragility syndrome with plakophilin-1 deficiency. This is the first occurrence of ectodermal dysplasia-skin fragility syndrome in an animal species. Controlled mating of carrier dogs would yield puppies that could, in theory, be tested for gene therapy of this rare but severe skin disease of children.

Expression of Plakophilins (PKP1, PKP2, and PKP3) in breast cancers.

UNLABELLED: Plakophilins (PKP) are desmosomal plague proteins, which belong to the p120ctn subfamily of armadillo repeat containing proteins. We aimed to analyze the role of plakophilins in breast cancer and its clinical progress. We have performed immunohistochemical study of the PKP1,2,3 in breast carcinoma. The study included 108 patients with breast cancer and 26 age- and sex-matched healthy controls. We investigated the associations between staining intensity and some clinicopathologic features like tumor size, axillary node status, stage, lymphovascular invasion, perineural invasion, grade, hormone receptor status, and c-erb B2. The mean age of patients was 46 years (22-78). In breast cancer, compared with normal tissue, PKP1 and PKP2 expressions were indifferent (P > 0.05), but PKP3 expression was significantly increased in breast cancer (P = 0.0014). Although PKP1 and PKP2 expression levels were not correlated with clinicopathological parameters, increased PKP3 expression was positively correlated with node positivity and grade (P = 0.000, P = 0.000). CONCLUSION: Overexpressed PKP3 is likely to be an essential contributor to a growth-promoting pathway and to aggressive features of breast cancer.

A Boyden chamber-based method for characterization of astrocyte protrusion localized RNA and protein.

The Boyden chamber assay has been developed for various cell migration and invasion protocols. One variant of the Boyden chamber assay is the pseudopodium isolation assay, which has been developed to identify RNA and proteins localized in pseudopodia cell protrusions. Astrocytes are the most abundant cell type in the CNS and typically extend long cellular protrusions. Increasing interest emerges concerning for example the growth mechanisms and functions of astrocytes in respect to brain development, re-uptake of neurotransmitters in the synaptic cleft and glial scar formation. Protein and RNA localization mechanisms have been extensively examined in neurons and shown to play pivotal roles for the functional presence of specific protein components in neuronal protrusions. Here we present a simple Boyden chamber based method to isolate astrocyte cell protrusions for biochemical analysis. We have succeeded in isolating protrusion localized RNA and protein from the mouse astrocyte cell line, C8-S, and mouse primary astrocytes. This is exemplified in biochemical analyses showing specific localization of the mRNA for Ras-related protein (Rab13), Plakophilin-4 (Pkp4), Ankyrin Repeat Domain 25 (Ankrd25), and inositol polyphosphate-1-phosphatase (Inpp1) in the protrusions of both C8-S cells and primary astrocytes. Concordant, the Pkp4 protein was also predominantly localized in the protrusions of C8-S and primary astrocytes. The described methodology can be the basis for both genome wide and specific descriptive and functional studies of RNA and protein localization in the protrusions of astrocytes which could contribute considerably to the existing knowledge of astrocyte functions in the CNS.

Relative contribution of changes in sodium current versus intercellular coupling on reentry initiation in 2-dimensional preparations of plakophilin-2-deficient cardiac cells.

BACKGROUND: Loss of expression of the desmosomal protein plakophilin-2 (PKP2) leads to decreased gap junction-mediated (GJ) coupling, and alters the amplitude and kinetics of sodium current in cardiac myocytes. Whether these modifications, alone or in combination, are sufficient to act as arrhythmogenic substrates remains undefined. OBJECTIVE: This study sought to characterize arrhythmia susceptibility and reentry dynamics consequent to loss of PKP2 expression, and to assess the relative contribution of cell uncoupling versus alterations in sodium current in generation of reentry. METHODS: Monolayers of neonatal rat ventricular myocytes were treated with oligonucleotides that either prevented or failed to prevent PKP2 expression. Numerical simulations modeled experimentally observed modifications in I(Na), GJ coupling, or both (models PKP2-Na, PKP2-GJ, and PKP2-KD, respectively). Relative roles of sodium current density versus kinetics were further explored. RESULTS: Loss of PKP2 expression increased incidence of rotors and decreased frequency of rotation. Mathematical simulations revealed that single premature stimuli initiated rotors in models PKP2-Na and PKP2-KD, but not PKP2-GJ. Changes in sodium current kinetics, rather than current density, were key to reentry initiation. Anatomical barriers led to vortex shedding, wavebreaks, and rotors when I(Na) kinetics, but not GJ coupling or I(Na) density, were altered. CONCLUSION: PKP2-dependent changes in sodium current kinetics lead to slow conduction, increased propensity to functional block, and vortex shedding. Changes in GJ or I(Na) density played only a minor role on reentry susceptibility. Changes in electrical properties of the myocyte caused by loss of expression of PKP2 can set the stage for rotors even if anatomical homogeneity is maintained.

Sex differences in expression and subcellular localization of heart rhythm determinant proteins.

To evaluate sex differences in protein expression in the heart, we performed Western blot studies on a subset of Heart Rhythm Determinant (HRD) proteins. We examined key components of a variety of types of mechanical and electrical junctions including, connexin43, plakophilin-2, N-cadherin and plakoglobin, ankyrin-2 and actin. We describe novel findings in sex differences in cardiac protein expression and membrane localization. For most proteins examined, sex differences were significantly more pronounced in the membrane compartment than in overall expression. These studies extend our previous findings in microarray studies to demonstrate that sex differences in gene expression are likely to confer distinct functional properties on male and female myocardium.

Cordial connections: molecular ensembles and structures of adhering junctions connecting interstitial cells of cardiac valves in situ and in cell culture.

Remarkable efforts have recently been made in the tissue engineering of heart valves to improve the results of valve transplantations and replacements, including the design of artificial valves. However, knowledge of the cell and molecular biology of valves and, specifically, of valvular interstitial cells (VICs) remains limited. Therefore, our aim has been to determine and localize the molecules forming the adhering junctions (AJs) that connect VICs in situ and in cell culture. Using biochemical and immunolocalization methods at the light- and electron-microscopic levels, we have identified, in man, cow, sheep and rat, the components of VIC-connecting AJs in situ and in cell culture. These AJs contain, in addition to the transmembrane glycoproteins N-cadherin and cadherin-11, the typical plaque proteins alpha- and beta-catenin as well as plakoglobin and p120, together with minor amounts of protein p0071, i.e. a total of five plaque proteins of the armadillo family. While we can exclude the occurrence of desmogleins, desmocollins and desmoplakin, we have noted with surprise that AJs of VICs in cell cultures, but not those growing in the valve tissue, contain substantial amounts of the desmosomal plaque protein, plakophilin-2. Clusters of AJs occur not only on the main VIC cell bodies but are also found widely dispersed on their long filopodia thus forming, in the tissue, a meshwork that, together with filopodial attachments to paracrystalline collagen fiber bundles, establishes a three-dimensional suprastructure, the role of which is discussed with respect to valve formation, regeneration and function.

Connexin43 remodeling caused by inhibition of plakophilin-2 expression in cardiac cells.

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.