Mutations In Desmosomal Proteins Research Articles

Chronic diastolic stretch unmasks conduction defects in an in vitro model of arrhythmogenic cardiomyopathy.

We seek to elucidate the precise nature of mechanical loading that precipitates conduction deficits in a concealed-phase model of arrhythmogenic cardiomyopathy (ACM). ACM is a progressive disorder often resulting from mutations in desmosomal proteins. Exercise has been shown to worsen disease progression and unmask arrhythmia vulnerability, yet the underlying pathomechanisms may depend on the type and intensity of exercise. Because exercise causes myriad changes to multiple inter-dependent hemodynamic parameters, it is difficult to isolate its effects to specific changes in mechanical load. Here, we use engineered heart tissues (EHTs) with iPSC-derived cardiomyocytes expressing R451G desmoplakin, an ACM-linked mutation, which results in a functionally null model of desmoplakin (DSP). We also use a novel bioreactor to independently perturb tissue strain at different time points during the cardiac cycle. We culture EHTs under three strain regimes: normal physiological shortening; increased diastolic stretch, simulating high preload; and isometric culture, simulating high afterload. DSPR451G EHTs that have been cultured isometrically undergo adaptation, with no change in action potential parameters, conduction velocity, or contractile function, a phenotype confirmed by global proteomic analysis. However, when DSPR451G EHTs are subjected to increased diastolic stretch, they exhibit concomitant reductions in conduction velocity and the expression of connexin-43. These effects are rescued by inhibition of both lysosome activity and ERK signaling. Our results indicate that the response of DSPR451G EHTs to mechanical stimuli depends on the strain and the timing of the applied stimulus, with increased diastolic stretch unmasking conduction deficits in a concealed-phase model of ACM.

Buccal mucosa cells as a diagnostic tool in patients with arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Netherlands Cardio Vascular Research Initiative (CVON): the Dutch heart foundation Background Arrhythmogenic cardiomyopathy (ACM) is predominantly caused by mutations in genes encoding desmosomal proteins (most often multiple different mutations in plakophilin-2), however also mutations in non-desmosomal proteins like phospholamban (PLN, PLNR14Del) are found. Previous investigations showed that plakoglobin protein levels and localization in cardiac tissue of ACM patients is disturbed and this could be a valuable additional tool to discriminate between non-affected family members and those being at risk during progression of the disease. Information about cardiac plakoglobin status can only be obtained via endocardial biopsies, however, this technique has major draw-backs and risks, and therefore an alternative is needed. A promising new non-invasive tool is the use of buccal mucosa cells (BMC), as it has been reported that effects of mutations in desmosomal proteins are additionally reflected in non-cardiac tissues like buccal mucosa smears that do also express those desmosomal genes. Purpose To investigate the clinical usability of BMC as tool to classify patients at risk of developing ACM by comparing healthy controls with asymptomatic and symptomatic classical ACM patients, and patients carrying a PLNR14Del mutation. Methods and results BMC of 84 human subjects, 33 ACM patients (19 males, 28 with a PKP2 mutation), 17 PLN patients (6 males) and 34 healthy controls (14 males), were collected on glass slides, fixed and labelled with anti-plakoglobin antibodies diluted 1:5.000, 1:10.000, 1:20.000 and 1:40.000, and scored for their membrane labelling. Data revealed that for each applied dilution, plakoglobin protein levels at the membrane were significantly reduced in BMC obtained from ACM patients compared to healthy controls. This effect appeared independent from age and sex of the patients. Furthermore, dilutions 1:5.000 and 1:10.000 showed a moderate correlation with the revised 2010 Task Force Criteria Score (TFC) which are commonly used for ACM diagnosis (Rs -0.67, n = 64, p <0.0001 and Rs -0.71, n = 64, p < 0.0001 resp.) In contrast, plakoglobin scores of PLN patients were similar to controls. Conclusion On the population level, there is a significant reduction of plakoglobin protein in BMC membranes of ACM patients but not for patients bearing the PLNR14Del mutation when compared to healthy controls. However, for clinical diagnosis of the individual patient, this method is most likely not discriminative enough to distinguish patients from healthy controls, because of the high interindividual variability.

Regulation of cardiac myocyte cohesion and gap junctions via desmosomal adhesion.

Mutations in desmosomal proteins can induce arrhythmogenic cardiomyopathy with life-threatening arrhythmia. Previous data demonstrated adrenergic signalling to be important to regulate desmosomal cohesion in cardiac myocytes. Here, we investigated how signalling pathways including adrenergic signalling, PKC and SERCA regulate desmosomal adhesion and how this controls gap junctions (GJs) in cardiac myocytes. Immunostaining, Western blot, dissociation assay and multi-electrode array were applied in HL-1 cardiac myocytes to evaluate localization, expression and function of desmosomal and GJ components. cAMP levels were determined by ELISA. Activation of PKC by PMA or adrenergic signalling increased cell cohesion and desmoglein-2 and desmoplakin localization at cell-cell junctions, whereas tryptophan (Trp) treatment to inhibit cadherin binding or inhibition of SERCA by thapsigargin reduced cell cohesion, while cAMP elevation rescued this effect. Despite no changes in protein expression, accumulation of GJ protein connexin-43 was detectable at cell-cell contacts in parallel to increased cohesion. Disruption of cell cohesion by Trp, PMA or thapsigargin impaired conduction of excitation comparable to GJ inhibition. cAMP elevation was effective to improve arrhythmia after Trp treatment. Weakened cell cohesion by Trp or depletion of desmoglein-2 or plakoglobin blocked signalling via the β1-adrenergic receptor. Moreover, silencing of desmosomal proteins increased arrhythmia and reduced conduction velocity, which were rescued by cAMP elevation. These data demonstrate the interplay of GJs, desmosomes and the β1-adrenergic receptor with regulation of their function by cell cohesion, adrenergic and PKC signalling or SERCA inhibition. These results support the identification of new targets to treat arrhythmogenic cardiomyopathy.

Mechanical loading of desmosomes depends on the\xa0magnitude and orientation of external stress

Desmosomes are intercellular adhesion complexes that connect the intermediate filament cytoskeletons of neighboring cells, and are essential for the mechanical integrity of mammalian tissues. Mutations in desmosomal proteins cause severe human pathologies including epithelial blistering and heart muscle dysfunction. However, direct evidence for their load-bearing nature is lacking. Here we develop Förster resonance energy transfer (FRET)-based tension sensors to measure the forces experienced by desmoplakin, an obligate desmosomal protein that links the desmosomal plaque to intermediate filaments. Our experiments reveal that desmoplakin does not experience significant tension under most conditions, but instead becomes mechanically loaded when cells are exposed to external mechanical stresses. Stress-induced loading of desmoplakin is transient and sensitive to the magnitude and orientation of the applied tissue deformation, consistent with a stress absorbing function for desmosomes that is distinct from previously analyzed cell adhesion complexes.

Genetics of and pathogenic mechanisms in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease, associated with a high risk of sudden cardiac death. ARVC has been termed a 'disease of the desmosome' based on the fact that in many cases, it is caused by mutations in genes encoding desmosomal proteins at the specialised intercellular junctions between cardiomyocytes, the intercalated discs. Desmosomes maintain the structural integrity of the ventricular myocardium and are also implicated in signal transduction pathways. Mutated desmosomal proteins are thought to cause detachment of cardiac myocytes by the loss of cellular adhesions and also affect signalling pathways, leading to cell death and substitution by fibrofatty adipocytic tissue. However, mutations in desmosomal proteins are not the sole cause for ARVC as mutations in non-desmosomal genes were also implicated in its pathogenesis. This review will consider the pathology, genetic basis and mechanisms of pathogenesis for ARVC.

Potential new mechanisms of pro-arrhythmia in arrhythmogenic cardiomyopathy: focus on calcium sensitive pathways.

Arrhythmogenic cardiomyopathy, or its most well-known subform arrhythmogenic right ventricular cardiomyopathy (ARVC), is a cardiac disease mainly characterised by a gradual replacement of the myocardial mass by fibrous and fatty tissue, leading to dilatation of the ventricular wall, arrhythmias and progression towards heart failure. ARVC is commonly regarded as a disease of the intercalated disk in which mutations in desmosomal proteins are an important causative factor. Interestingly, the Dutch founder mutation PLN R14Del has been identified to play an additional, and major, role in ARVC patients within the Netherlands. This is remarkable since the phospholamban (PLN) protein plays a leading role in regulation of the sarcoplasmic reticulum calcium load rather than in the establishment of intercellular integrity. In this review we outline the intracellular cardiac calcium dynamics and relate pathophysiological signalling, induced by disturbed calcium handling, with activation of calmodulin dependent kinase II (CaMKII) and calcineurin A (CnA). We postulate a thus far unrecognised role for Ca2+ sensitive signalling proteins in maladaptive remodelling of the macromolecular protein complex that forms the intercalated disk, during pro-arrhythmic remodelling of the heart.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy-three decades of progress.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a rare, inherited cardiomyopathy characterized by ventricular arrhythmias, sudden cardiac death, and right ventricular dysfunction. Since the first major description of this disease, much has been learned about ARVD/C. One of the main breakthroughs was the discovery that mutations in desmosomal proteins are the most frequent genetic basis of ARVD/C. Today, genetic testing plays an important role in both the diagnosis of ARVD/C and cascade family screening. Much has also been learned concerning the optimal approaches to diagnosis. The 2010 Task Force Diagnostic criteria for ARVD/C represent the standard for diagnosis today. We have also learned much about the importance of proband status and the 24-h PVC count to assess sudden death risk, and the importance of exercise both in the development of ARVD/C in susceptible individuals and in defining the course of the disease. From a treatment perspective, placement of ICDs in specific subsets of patients with ARVD/C who are at increased risk of sudden death is important. The techniques of VT ablation have also evolved over time and are valuable components of our management strategies for the ARVD/C patient today. This review will provide an update on ARVD/C, with specific attention to some of the contributions to this field reported by the Johns Hopkins ARVD/C Program.

205 Atrial Arrhythmia Susceptibility in Arrhythmogenic Right Ventricular Cardiomyopathy

BackgroundArrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes sudden death and right ventricular heart failure in young athletes. Loss-of-function mutations in desmosomal proteins including plakoglobin deficiency are...

Mutation in KANK2, encoding a sequestering protein for steroid receptor coactivators, causes keratoderma and woolly hair

BackgroundThe combination of palmoplantar keratoderma and woolly hair is uncommon and reported as part of Naxos and Carvajal syndromes, both caused by mutations in desmosomal proteins and associated with cardiomyopathy....

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study

Background:Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC.Methods and Results:We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3). Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed.The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% ± 0.3% in controls, vs. 4.6% ± 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% ± 0.3% in controls vs. 4.4% ± 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 ± 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1). For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease. Conclusions:The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis.

Eliminating Ventricular Tachycardia by Targeting Premature Ventricular Contractions in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an electrophysiological curiosity. Unlike most forms of ventricular tachycardia (VT) due to a structural pathogenesis, it commonly originates from the right ventricle (RV) rather than the left ventricle, and in contrast to most forms of malignant ventricular arrhythmias due to a molecular defect, ARVD/C develops from mutations in desmosomal proteins rather than ion channels. Therefore, ARVD/C stands alone as an example of a cardiomyopathy, which is expressed primarily in the RV and is associated with VT and sudden cardiac death. Article see p 160 In general, ARVD/C is an autosomal dominant disease. The precise correlation between mutations in desmosomal proteins and disease expression is unclear, but desmosomal proteins are integral to cell adhesion, are involved with transcriptional regulation of genes that govern adipogenesis and apoptosis, and facilitate electrical conductivity between cells through regulation of gap junctions.1 Pathologically, there is a progressive loss of cardiomyocytes with fibrofatty replacement. Although initially confined to the RV, and in particular, to the RV inflow tract, the RV outflow tract and apex, progression of fibrofatty replacement in the remainder of the RV and left ventricle may occur. This process begins in the subepicardium and midmyocardium, and subsequently migrates to the subendocardium. Because of the presence of patchy scar interspersed with normal myocardium, the demonstration of entrainment during tachycardia, as well as the presence of anatomic barriers like the tricuspid annulus, re-entry is well established as the arrhythmogenic mechanism of VT in ARVD/C.2 Most, but not all, of these circuits are due to macroreentry, with a minority due to focal re-entry.3,4 The study by Philips et al5 in this issue of the journal suggests that activation mapping and pace mapping of frequent premature ventricular contractions (PVCs) can identify focal sites where successful ablation of …

Desmosome–Ion Channel Interactions and Their Possible Role in Arrhythmogenic Cardiomyopathy

Most commonly, arrhythmogenic cardiomyopathy (also known as arrhythmogenic right ventricular cardiomyopathy, or ARVC) is caused by mutations in desmosomal proteins. The question arises as to the mechanisms by which mutations in mechanical junctions, affect the rhythm of the heart. We have proposed that a component of the arrhythmogenic substrate may include changes in the function of both, gap junctions and sodium channels. Here, we review the relevant literature on this subject.

Mouse Models in Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heart muscle disorder characterized by fibro-fatty replacement of cardiomyocytes. The cardinal manifestations are arrhythmias, sudden cardiac death, and seldom heart failure. Mutations in genes encoding desmosomal proteins and their interaction partners have been implicated in the pathogenesis of ARVC and it is now widely accepted that ARVC is a disease caused by abnormal cell–cell adhesion. The mechanism(s) by which mutations in desmosomal proteins lead to fibro-fatty replacement remains to be fully elucidated. To this aim over the last 10 years different transgenic and targeted mouse models have been developed, these models and what they have taught us will be discussed in this review.

Novel plakophilin2 mutation: Three-generation family with arrhythmogenic right ventricular cardiomyopathy

Objectives. The autosomal dominant form of arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins. A mutation in plakophilin 2 (PKP 2) is a frequent cause for ARVC. We describe a new mutation in the PKP2 gene, the genotype-phenotype variation in this mutation and its clinical consequences. Design. Individuals in a three-generation family were investigated after the sudden cardiac death of a young male. Clinical evaluation, electrocardiography, echocardiography, magnetic resonance imaging, endomyocardial w. Results. A novel heterozygote mutation, a c.368G > A transition, located in exon 3 of the PKP2 gene was found (p.Trp123X). The phenotype was characterized by arrhythmia at an early age in some individuals, with mild abnormalities on imaging. Conclusions. This new plakophilin mutation demonstrates variable penetrance and phenotypic expression in ARVC, and highlights the need of genetic testing and thorough phenotype examination in ARVC pedigrees.

Desmoglein 2 mutant mice develop cardiac fibrosis and dilation

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-011-0175-y) contains supplementary material, which is available to authorized users.

Arrhythmogenic right ventricular cardiomyopathy: new insights into mechanisms of disease

Arrhythmogenic right ventricular cardiomyopathy is a primary heart muscle disorder characterized by the early occurrence of arrhythmias often out of proportion to the extent of structural remodeling and contractile derangement. Approximately 40% of patients with arrhythmogenic right ventricular cardiomyopathy have one or more mutations in genes encoding proteins in desmosomes, intercellular adhesion junctions which, in cardiac myocytes, reside within intercalated disks. Some desmosomal proteins fulfill roles both as structural proteins in cell-cell adhesion junctions and as signaling molecules in pathways mediated by Wnt ligands. Evidence is increasing that mutations in desmosomal proteins can perturb the normal balance of critical proteins in junctions and the cytosol which, in turn, could alter gene expression by circumventing normal Wnt signaling pathways. This review highlights recent advances in understanding the pathogenesis of arrhythmogenic right ventricular cardiomyopathy and presents evidence suggesting that the disease is caused by a combination of altered cellular biomechanical behavior and altered signaling.

Multiple mutations in desmosomal proteins encoding genes in arrhythmogenic right ventricular cardiomyopathy/dysplasia

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is a progressive cardiomyopathy showing a wide clinical spectrum in terms of clinical expressions and prognoses. This study sought to estimate the occurrence of compound and double heterozygotes for mutations in desmosomal proteins encoding genes in a cohort of ARVC/D Italian index cases, and to assess the clinical phenotype of mutations carriers. Fourty-two consecutive ARVC/D index cases who fulfilled the International Task Force diagnostic criteria were screened for mutations in PKP2, DSP, DSG2, DSC2, and JUP genes by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Three probands (7.1%) showing a family history of sudden death carried multiple mutations. Family screening identified an additional 7 multiple-mutation carriers. Among the 7 double heterozygotes for mutations in different genes, 2 were clinically unaffected, 2 were affected, and 3 showed some clinical signs of ARVC/D even if they did not fulfill the diagnostic criteria. Two compound heterozygotes for mutations in the same gene and 1 subject carrying 3 different mutations showed a severe form of the disease with heart failure onset at a young age. Moreover, multiple-mutation carriers showed a higher prevalence of left ventricular involvement (P = .025) than single-mutation carriers. Occurrence of compound and double heterozygotes in ARVC/D index cases is particularly relevant to mutation screening strategy and to genetic counseling. Even if multiple-mutation carriers show a wide variability in clinical expression, the extent of the disease is higher compared to that in single-mutation carriers.

Skin and heart: une liaison dangereuse

Both skin and heart are subject to shear mechanical stress and need to be stress-resistant in a flexible way. The intercellular connecting structures in skin and heart, the desmosomes, that have to resist these forces show remarkable resemblance in epidermis and myocardium. Mutations in desmosomal proteins lead to inherited desmosomal cardiocutaneous syndromes (DCCS): une liaison dangereuse. This article will critically review the cutaneous and cardiac features as well as the molecular background of DCCS, such as Naxos disease and Carvajal syndrome caused by deficiencies of plakoglobin and desmoplakin respectively. In addition, potential other desmosomal gene candidates for an involvement in cardiocutaneous syndromes are considered. The skin features in these syndromes may be the hallmark for the presence of progressive and ultimately lethal cardiac disease. Knowledge of these skin features and early recognition of such a syndrome may provide opportunities to halt or slow down cardiac disease progression, treat arrhythmias and even prevent sudden death.