Atrial cardiomyopathy resulting from loss of plakophilin-2 expression: Response to adrenergic stimulation and implications for the exercise response.

Atrial arrhythmias occur in 20-40% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) and are associated with an increased risk of sustained ventricular arrhythmias and inappropriate implantable cardioverter-defibrillator shocks. The pathophysiology of atrial arrhythmias in ARVC remains unclear. Most cases of gene-positive ARVC are linked to pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Here, we test the hypothesis that loss of PKP2 expression leads to pro-arrhythmic changes in atrial cardiomyocytes. Atrial cells/tissue were obtained from a cardiac-specific, tamoxifen-activated model of PKP2 deficiency (PKP2cKO). By contrast to PKP2cKO ventricular myocytes, PKP2cKO atrial cardiomyocytes presented no significant differences in intracellular calcium (Ca2+ i) transient dynamics, sarcoplasmic reticulum load or action potential morphology. PKP2cKO atrial cardiomyocytes showed elevated reactive oxygen species levels, increased frequency and amplitude of Ca2+ sparks, and increased diastolic [Ca2+]i compared to control; the latter two parameters were further increased by isoproterenol exposure and reversed by exposure to ryanodine receptor blocker dantrolene. We speculate that these isoproterenol-dependent effects may impact on the exercise-related atrial arrhythmia risk in ARVC patients. Despite absence of changes in Ca2+ i transient dynamics, PKP2cKO atrial cardiomyocytes showed enhanced sarcomere shortening and impaired sarcomere relaxation. Orthogonal transcriptomic analysis of human(GTEx) and PKP2cKO atrial tissue led to identification of 41 transcripts depending on PKP2 expression. Biochemical follow-up confirmed reduced abundance of sarcomeric protein myosin binding protein C, potentially playing a role in cellular shortening and relaxation changes observed. Our findings provide novel insights into the role of PKP2 in atrial myocardium with potential implications to therapeutic management of atrial fibrillation in patients with PKP2-related ARVC. KEY POINTS: Atrial arrhythmias occur in a large group of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a cardiac disease mostly caused by pathogenic variants in the desmosomal gene plakophilin-2 (PKP2). Exercise is considered to be an independent risk factor for arrhythmias consequent to PKP2 deficiency. We show that loss of PKP2 expression affects cellular calcium handling and electrophysiology differently in left atrial vs. ventricular myocardium and causes extensive atrial fibrosis. PKP2-deficient atrial cardiomyocytes present increased spontaneous sarcoplasmic reticulum calcium release events, further enhanced by isoproterenol exposure and reversible by a ryanodine receptor blocker (dantrolene). In addition, PKP2-deficient atrial myocytes exhibit impaired relaxation and enhanced sarcomere shortening, most probably related to reduced abundance of myosin binding protein C. We speculate that cellular effects reported upon isoproterenol impact on the exercise-related atrial arrhythmia risk in ARVC patients. We further propose that therapeutic approaches aimed at mitigating ventricular damage may be effective to treat the atrial disease in ARVC.

Body surface potential mapping detects early disease onset in plakophilin-2-pathogenic variant carriers.

AIMS: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a progressive inherited cardiac disease. Early detection of disease and risk stratification remain challenging due to heterogeneous phenotypic expression. The standard configuration of the 12 lead electrocardiogram (ECG) might be insensitive to identify subtle ECG abnormalities. We hypothesized that body surface potential mapping (BSPM) may be more sensitive to detect subtle ECG abnormalities. METHODS AND RESULTS: We obtained 67 electrode BSPM in plakophilin-2 (PKP2)-pathogenic variant carriers and control subjects. Subject-specific computed tomography/magnetic resonance imaging based models of the heart/torso and electrode positions were created. Cardiac activation and recovery patterns were visualized with QRS- and STT-isopotential map series on subject-specific geometries to relate QRS-/STT-patterns to cardiac anatomy and electrode positions. To detect early signs of functional/structural heart disease, we also obtained right ventricular (RV) echocardiographic deformation imaging. Body surface potential mapping was obtained in 25 controls and 42 PKP2-pathogenic variant carriers. We identified five distinct abnormal QRS-patterns and four distinct abnormal STT-patterns in the isopotential map series of 31/42 variant carriers. Of these 31 variant carriers, 17 showed no depolarization or repolarization abnormalities in the 12 lead ECG. Of the 19 pre-clinical variant carriers, 12 had normal RV-deformation patterns, while 7/12 showed abnormal QRS- and/or STT-patterns. CONCLUSION: Assessing depolarization and repolarization by BSPM may help in the quest for early detection of disease in variant carriers since abnormal QRS- and/or STT-patterns were found in variant carriers with a normal 12 lead ECG. Because electrical abnormalities were observed in subjects with normal RV-deformation patterns, we hypothesize that electrical abnormalities develop prior to functional/structural abnormalities in ARVC.

Myocarditis as a trigger for the expression of biventricular arrhythmogenic cardiomyopathy in desmosomal gene mutation.

Arrhythmogenic-cardiomyopathy (ACM) is an inherited heart disease with right, left, or biventricular (BVACM) involvement based on EKG, imaging, family history, and genetic testing. We present a 64-year-old woman with prior myocarditis and diagnosis of BVACM 29 years later. We propose myocarditis as a promoter of gene expression of plakophilin-2 mutation.

Role of plakophilin-2 expression on exercise-related progression of arrhythmogenic right ventricular cardiomyopathy: a translational study.

AIMS: Exercise increases arrhythmia risk and cardiomyopathy progression in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients, but the mechanisms remain unknown. We investigated transcriptomic changes caused by endurance training in mice deficient in plakophilin-2 (PKP2cKO), a desmosomal protein important for intercalated disc formation, commonly mutated in ARVC and controls. METHODS AND RESULTS: Exercise alone caused transcriptional downregulation of genes coding intercalated disk proteins. The changes converged with those in sedentary and in exercised PKP2cKO mice. PKP2 loss caused cardiac contractile deficit, decreased muscle mass and increased functional/transcriptomic signatures of apoptosis, despite increased fractional shortening and calcium transient amplitude in single myocytes. Exercise accelerated cardiac dysfunction, an effect dampened by pre-training animals prior to PKP2-KO. Consistent with PKP2-dependent muscle mass deficit, cardiac dimensions in human athletes carrying PKP2 mutations were reduced, compared to matched controls. CONCLUSIONS: We speculate that exercise challenges a cardiomyocyte "desmosomal reserve" which, if impaired genetically (e.g., PKP2 loss), accelerates progression of cardiomyopathy.

The arrhythmogenic cardiomyopathy phenotype associated with PKP2 c.1211dup variant.

BACKGROUND: The arrhythmogenic cardiomyopathy (ACM) phenotype, with life-threatening ventricular arrhythmias and heart failure, varies according to genetic aetiology. We aimed to characterise the phenotype associated with the variant c.1211dup (p.Val406Serfs*4) in the plakophilin­2 gene (PKP2) and compare it with previously reported Dutch PKP2 founder variants. METHODS: Clinical data were collected retrospectively from medical records of 106 PKP2 c.1211dup heterozygous carriers. Using data from the Netherlands ACM Registry, c.1211dup was compared with 3 other truncating PKP2 variants (c.235C > T (p.Arg79*), c.397C > T (p.Gln133*) and c.2489+1G > A (p.?)). RESULTS: Of the 106 carriers, 47 (44%) were diagnosed with ACM, at a mean age of 41 years. By the end of follow-up, 29 (27%) had experienced sustained ventricular arrhythmias and 12 (11%) had developed heart failure, with male carriers showing significantly higher risks than females on these endpoints (p < 0.05). Based on available cardiac magnetic resonance imaging and echocardiographic data, 46% of the carriers showed either right ventricular dilatation and/or dysfunction, whereas a substantial minority (37%) had some form of left ventricular involvement. Both geographical distribution of carriers and haplotype analysis suggested PKP2 c.1211dup to be a founder variant originating from the South-Western coast of the Netherlands. Finally, a Cox proportional hazards model suggested significant differences in ventricular arrhythmia-free survival between 4 PKP2 founder variants, including c.1211dup. CONCLUSIONS: The PKP2 c.1211dup variant is a Dutch founder variant associated with a typical right-dominant ACM phenotype, but also left ventricular involvement, and a possibly more severe phenotype than other Dutch PKP2 founder variants.

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.

Genetic Variant Score and Arrhythmogenic Right Ventricular Cardiomyopathy Phenotype in Plakophilin-2 Mutation Carriers.

INTRODUCTION: Whether detailed genetic information contributes to risk stratification of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) remains uncertain. Pathogenic genetic variants in some genes seem to carry a higher risk for arrhythmia and earlier disease onset than others, but comparisons between variants in the same gene have not been done. Combined Annotation Dependent Depletion (CADD) score is a bioinformatics tool that measures the pathogenicity of each genetic variant. We hypothesized that a higher CADD score is associated with arrhythmic events and earlier age at ARVC manifestations in individuals carrying pathogenic or likely pathogenic genetic variants in plakophilin-2 (PKP2). METHODS: CADD scores were calculated using the data from pooled Scandinavian and North American ARVC cohorts, and their association with cardiac events defined as ventricular tachycardia/ventricular fibrillation (VT/VF) or syncope and age at definite ARVC diagnosis were assessed. RESULTS: In total, 33 unique genetic variants were reported in 179 patients (90 males, 71 probands, 96 with definite ARVC diagnosis at a median age of 35 years). Cardiac events were reported in 76 individuals (43%), of whom 53 had sustained VT/VF (35%). The CADD score was neither associated with age at cardiac events (HR 1.002, 95% CI: 0.953-1.054, p = 0.933) nor with age at definite ARVC diagnosis (HR 0.992, 95% CI: 0.947-1.039, p = 0.731). CONCLUSION: No correlation was found between CADD scores and clinical manifestations of ARVC, indicating that the score has no additional risk stratification value among carriers of pathogenic or likely pathogenic PKP2 genetic variants.

Risk stratification and subclinical phenotyping of dilated and/or arrhythmogenic cardiomyopathy mutation-positive relatives: CVON eDETECT consortium.

In relatives of index patients with dilated cardiomyopathy and arrhythmogenic cardiomyopathy, early detection of disease onset is essential to prevent sudden cardiac death and facilitate early treatment of heart failure. However, the optimal screening interval and combination of diagnostic techniques are unknown. The clinical course of disease in index patients and their relatives is variable due to incomplete and age-dependent penetrance. Several biomarkers, electrocardiographic and imaging (echocardiographic deformation imaging and cardiac magnetic resonance imaging) techniques are promising non-invasive methods for detection of subclinical cardiomyopathy. However, these techniques need optimisation and integration into clinical practice. Furthermore, determining the optimal interval and intensity of cascade screening may require a personalised approach. To address this, the CVON-eDETECT (early detection of disease in cardiomyopathy mutation carriers) consortium aims to integrate electronic health record data from long-term follow-up, diagnostic data sets, tissue and plasma samples in a multidisciplinary biobank environment to provide personalised risk stratification for heart failure and sudden cardiac death. Adequate risk stratification may lead to personalised screening, treatment and optimal timing of implantable cardioverter defibrillator implantation. In this article, we describe non-invasive diagnostic techniques used for detection of subclinical disease in relatives of index patients with dilated cardiomyopathy and arrhythmogenic cardiomyopathy.

Disruption of Ca2+i Homeostasis and Connexin 43 Hemichannel Function in the Right Ventricle Precedes Overt Arrhythmogenic Cardiomyopathy in Plakophilin-2-Deficient Mice.

BACKGROUND: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+ transients, increased Ca2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca2+, increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca2+]i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. CONCLUSIONS: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Cardiac-Specific Caveolin-3 Overexpression Prevents Post-Myocardial Infarction Ventricular Arrhythmias by Inhibiting Ryanodine Receptor-2 Hyperphosphorylation.

INTRODUCTION: Ventricular arrhythmia is the most important risk factor for sudden cardiac death (SCD) after acute myocardial infarction (MI) worldwide. However, the molecular mechanisms underlying these arrhythmias are complex and not completely understood. OBJECTIVE: Here, we evaluated whether caveolin-3 (Cav3), the structural protein of caveolae, plays an important role in the therapeutic strategy for ventricular arrhythmias. METHODS: A model of cardiac-specific overexpression of Cav3 was established to evaluate the incidence of ventricular arrhythmias after MI in mice. Ca2+ imaging was employed to detect the propensity of adult murine cardiomyocytes to generate arrhythmias, and immunoprecipitation and immunofluorescence were used to determine the relationship of proteins. Additionally, qRT-PCR and western blotting were used to detect the mRNA and protein expression. RESULTS: We found that cardiac-specific overexpression of Cav3 delivered by a recombinant adeno-associated viral vector reduced the incidence of ventricular arrhythmias and SCD after MI in mice. Ca2+ imaging and western blotting revealed that overexpression of Cav3 reduced diastolic spontaneous Ca2+ waves by inhibiting the hyperphosphorylation of ryanodine receptor-2 (RyR2) at Ser2814, rather than at Ser2808, compared to in rAAV-red fluorescent protein control mice. Furthermore, we demonstrated that Cav3-regulated RYR2 hyperphosphorylation relied on plakophilin-2 in hypoxia-stimulated cultured cardiomyocytes by western blotting, immunoprecipitation, and immunofluorescence in vitro. CONCLUSIONS: Our results suggested a novel role for Cav3 in the prevention of ventricular arrhythmias, thereby identifying a new target for preventing SCD after MI.

Late presentation of arrhythmogenic right ventricular cardiomyopathy in an octogenarian associated with a pathogenic variant in the plakophilin 2 gene: a case report.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease characterized by fibrofatty replacement and ventricular arrhythmias. ARVC is believed to be a disease of the young, with most cases being diagnosed before the age of 40 years. We report here a case of newly diagnosed ARVC in an octogenarian associated with a pathogenic variant in the plakophilin 2 gene (PKP2). CASE PRESENTATION: An 80-year-old Japanese man was referred for sustained ventricular tachycardia. His baseline electrocardiogram showed negative T waves in V1-V4. Right ventriculography showed right ventricular aneurysm. Because this case met three major criteria, ARVC was diagnosed. He was successfully treated with radiofrequency ablation and oral amiodarone. Genetic analysis identified an insertion mutation in exon 8 of PKP2 (1725_1728dupGATG), which caused a frameshift and premature termination of translation (R577DfsX5). CONCLUSIONS: To the best of our knowledge, this is the first report of newly diagnosed ARVC in an octogenarian associated with a loss-of-function PKP2 pathogenic variant. Although the late clinical presentation of ARVC is rare, it should be included in the differential diagnosis when treating older patients with ventricular tachyarrhythmias.

Plakophilin-2 Haploinsufficiency Causes Calcium Handling Deficits and Modulates the Cardiac Response Towards Stress.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca2+ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca2+-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca2+ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

Calcium as a Key Player in Arrhythmogenic Cardiomyopathy: Adhesion Disorder or Intracellular Alteration?

Arrhythmogenic cardiomyopathy (ACM) is an inherited heart disease characterized by sudden death in young people and featured by fibro-adipose myocardium replacement, malignant arrhythmias, and heart failure. To date, no etiological therapies are available. Mutations in desmosomal genes cause abnormal mechanical coupling, trigger pro-apoptotic signaling pathways, and induce fibro-adipose replacement. Here, we discuss the hypothesis that the ACM causative mechanism involves a defect in the expression and/or activity of the cardiac Ca2+ handling machinery, focusing on the available data supporting this hypothesis. The Ca2+ toolkit is heavily remodeled in cardiomyocytes derived from a mouse model of ACM defective of the desmosomal protein plakophilin-2. Furthermore, ACM-related mutations were found in genes encoding for proteins involved in excitation‒contraction coupling, e.g., type 2 ryanodine receptor and phospholamban. As a consequence, the sarcoplasmic reticulum becomes more eager to release Ca2+, thereby inducing delayed afterdepolarizations and impairing cardiac contractility. These data are supported by preliminary observations from patient induced pluripotent stem-cell-derived cardiomyocytes. Assessing the involvement of Ca2+ signaling in the pathogenesis of ACM could be beneficial in the treatment of this life-threatening disease.

The human PKP2/plakophilin-2 gene is induced by Wnt/ß-catenin in normal and colon cancer-associated fibroblasts.

Colorectal cancer results from the malignant transformation of colonic epithelial cells. Stromal fibroblasts are the main component of the tumour microenvironment, and play an important role in the progression of this and other neoplasias. Wnt/ß-catenin signalling is essential for colon homeostasis, but aberrant, constitutive activation of this pathway is a hallmark of colorectal cancer. Here we present the first transcriptomic study on the effect of a Wnt factor on human colonic myofibroblasts. Wnt3A regulates the expression of 1,136 genes, of which 662 are upregulated and 474 are downregulated in CCD-18Co cells. A set of genes encoding inhibitors of the Wnt/ß-catenin pathway stand out among those induced by Wnt3A, which suggests that there is a feedback inhibitory mechanism. We also show that the PKP2 gene encoding the desmosomal protein Plakophilin-2 is a novel direct transcriptional target of Wnt/ß-catenin in normal and colon cancer-associated fibroblasts. PKP2 is induced by ß-catenin/TCF through three binding sites in the gene promoter and one additional binding site located in an enhancer 20 kb upstream from the transcription start site. Moreover, Plakophilin-2 antagonizes Wnt/ß-catenin transcriptional activity in HEK-293T cells, which suggests that it may act as an intracellular inhibitor of the Wnt/ß-catenin pathway. Our results demonstrate that stromal fibroblasts respond to canonical Wnt signalling and that Plakophilin-2 plays a role in the feedback control of this effect suggesting that the response to Wnt factors in the stroma may modulate Wnt activity in the tumour cells.

The Cardiac Sodium Channel and Its Protein Partners.

Activation of the electrical signal and its transmission as a depolarizing wave in the whole heart requires highly organized myocyte architecture and cell-cell contacts. In addition, complex trafficking and anchoring intracellular machineries regulate the proper surface expression of channels and their targeting to distinct membrane domains. An increasing list of proteins, lipids, and second messengers can contribute to the normal targeting of ion channels in cardiac myocytes. However, their precise roles in the electrophysiology of the heart are far from been extensively understood. Nowadays, much effort in the field focuses on understanding the mechanisms that regulate ion channel targeting to sarcolemma microdomains and their organization into macromolecular complexes. The purpose of the present section is to provide an overview of the characterized partners of the main cardiac sodium channel, NaV1.5, involved in regulating the functional expression of this channel both in terms of trafficking and targeting into microdomains.

Autosomal Recessive Nonsyndromic Arrhythmogenic Right Ventricular Cardiomyopathy without Cutaneous Involvements: A Novel Mutation.

The arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a genetic disease frequently associated with desmosomal mutations, mainly attributed to dominant mutations in the Plakophilin-2 (PKP2) gene. Naxos and Carvajal are the syndromic forms of ARVD/C due to recessive mutations. Herein, we report an autosomal recessive form of nonsyndromic ARVD/C caused by a mutation in the PKP2 gene. After examination and implementation of diagnostic modalities, the definite diagnosis of ARVD/C was confirmed by detection of ventricular tachycardia with a left bundle branch configuration and a superior axis, T-wave inversion in right precordial leads (i.e., V1-V3) in a 12-lead electrocardiogram, and a right ventricle outflow tract dilatation. Neither cutaneous involvement nor other abnormalities were observed. Genetic testing was performed during which an intronic mutation of c.2577+1G>T in the PKP2 gene was observed homozygously. The c.2577+1G>T disrupts PKP2 mRNA splicing and causes a nonsyndromic form of ARVD/C.

Identification of established arrhythmogenic right ventricular cardiomyopathy mutation in a patient with the contrasting phenotype of hypertrophic cardiomyopathy.

BACKGROUND: Advances in the nucleic acid sequencing technologies have ushered in the era of genetic-based "precision medicine". Applications of the genetic discoveries to practice of medicine, however, are hindered by phenotypic variability of the genetic variants. The report illustrates extreme pleiotropic phenotypes associated with an established causal mutation for hereditary cardiomyopathy. CASE PRESENTATION: We report a 61-year old white female who presented with syncope and echocardiographic and cardiac magnetic resonance (CMR) imaging findings consistent with the diagnosis of hypertrophic cardiomyopathy (HCM). The electrocardiogram, however, showed a QRS pattern resembling an Epsilon wave, a feature of arrhythmogenic right ventricular cardiomyopathy (ARVC). Whole exome sequencing (mean depth of coverage of exons 178X) analysis did not identify a pathogenic variant in the known HCM genes but identified an established causal mutation for ARVC. The mutation involves a canonical splice accepter site (c.2146-1G > C) in the PKP2 gene, which encodes plakophillin 2. Sanger sequencing confirmed the mutation. PKP2 is the most common causal gene for ARVC but has not been implicated in HCM. Findings on echocardiography and CMR during the course of 4-year follow up showed septal hypertrophy and a hyperdynamic left ventricle, consistent with the diagnosis of HCM. However, neither baseline nor follow up echocardiography and CMR studies showed evidence of ARVC. The right ventricle was normal in size, thickness, and function and there was no evidence of fibro-fatty infiltration in the myocardium. CONCLUSIONS: The patient carries an established pathogenic mutation for ARVC and a subtle finding of ARVC but exhibits the classic phenotype of HCM, a contrasting phenotype to ARVC. The case illustrates the need for detailed phenotypic characterization for patients with hereditary cardiomyopathies as well as the challenges physicians face in applying the genetic discoveries in practicing genetic-based "precision medicine".

Cardiac mesenchymal stromal cells are a source of adipocytes in arrhythmogenic cardiomyopathy.

AIM: Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. METHODS AND RESULTS: We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. CONTROLS: Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. CONCLUSIONS: Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies.

Multiple regulatory variants located in cell type-specific enhancers within the PKP2 locus form major risk and protective haplotypes for canine atopic dermatitis in German shepherd dogs.

BACKGROUND: Canine atopic dermatitis (CAD) is a chronic inflammatory skin disease triggered by allergic reactions involving IgE antibodies directed towards environmental allergens. We previously identified a ~1.5 Mb locus on canine chromosome 27 associated with CAD in German shepherd dogs (GSDs). Fine-mapping indicated association closest to the PKP2 gene encoding plakophilin 2. RESULTS: Additional genotyping and association analyses in GSDs combined with control dogs from five breeds with low-risk for CAD revealed the top SNP 27:19,086,778 (p = 1.4 × 10(-7)) and a rare ~48 kb risk haplotype overlapping the PKP2 gene and shared only with other high-risk CAD breeds. We selected altogether nine SNPs (four top-associated in GSDs and five within the ~48 kb risk haplotype) that spanned ~280 kb forming one risk haplotype carried by 35 % of the GSD cases and 10 % of the GSD controls (OR = 5.1, p = 5.9 × 10(-5)), and another haplotype present in 85 % of the GSD cases and 98 % of the GSD controls and conferring a protective effect against CAD in GSDs (OR = 0.14, p = 0.0032). Eight of these SNPs were analyzed for transcriptional regulation using reporter assays where all tested regions exerted regulatory effects on transcription in epithelial and/or immune cell lines, and seven SNPs showed allelic differences. The DNA fragment with the top-associated SNP 27:19,086,778 displayed the highest activity in keratinocytes with 11-fold induction of transcription by the risk allele versus 8-fold by the control allele (pdifference = 0.003), and also mapped close (~3 kb) to an ENCODE skin-specific enhancer region. CONCLUSIONS: Our experiments indicate that multiple CAD-associated genetic variants located in cell type-specific enhancers are involved in gene regulation in different cells and tissues. No single causative variant alone, but rather multiple variants combined in a risk haplotype likely contribute to an altered expression of the PKP2 gene, and possibly nearby genes, in immune and epithelial cells, and predispose GSDs to CAD.

Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.

BACKGROUND: Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy. METHODS AND RESULTS: We searched for PKP2 variants in the genomic DNA of 200 patients with a BrS diagnosis, no signs of arrhythmogenic cardiomyopathy, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at the site of cell contact. These deficits were restored by the transfection of wild-type PKP2, but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes from a patient with a PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of wild type, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to the reduced number of channels at the intercalated disc and increased separation of microtubules from the cell end. CONCLUSIONS: This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.