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Background: Autoimmunity is increasingly recognized as a key contributing factor in heart muscle diseases. The functional features of cardiac autoimmunity in humans remain undefined because of the challenge of studying immune responses in situ. We previously described a subset of c-mesenchymal epithelial transition factor (c-Met)–expressing (c-Met + ) memory T lymphocytes that preferentially migrate to cardiac tissue in mice and humans. Methods: In-depth phenotyping of peripheral blood T cells, including c-Met + T cells, was undertaken in groups of patients with inflammatory and noninflammatory cardiomyopathies, patients with noncardiac autoimmunity, and healthy controls. Validation studies were carried out using human cardiac tissue and in an experimental model of cardiac inflammation. Results: We show that c-Met + T cells are selectively increased in the circulation and in the myocardium of patients with inflammatory cardiomyopathies. The phenotype and function of c-Met + T cells are distinct from those of c-Met–negative (c-Met − ) T cells, including preferential proliferation to cardiac myosin and coproduction of multiple cytokines (interleukin-4, interleukin-17, and interleukin-22). Furthermore, circulating c-Met + T cell subpopulations in different heart muscle diseases identify distinct and overlapping mechanisms of heart inflammation. In experimental autoimmune myocarditis, elevations in autoantigen-specific c-Met + T cells in peripheral blood mark the loss of immune tolerance to the heart. Disease development can be halted by pharmacologic c-Met inhibition, indicating a causative role for c-Met + T cells. Conclusions: Our study demonstrates that the detection of circulating c-Met + T cells may have use in the diagnosis and monitoring of adaptive cardiac inflammation and definition of new targets for therapeutic intervention when cardiac autoimmunity causes or contributes to progressive cardiac injury.

Arrhythmogenic cardiomyopathy (ACM) is a primary disease of the myocardium, predominantly caused by genetic defects in proteins of the cardiac intercalated disc, particularly, desmosomes. Transmission is mostly autosomal dominant with incomplete penetrance. ACM also has wide phenotype variability, ranging from premature ventricular contractions to sudden cardiac death and heart failure. Among other drivers and modulators of phenotype, inflammation in response to viral infection and immune triggers have been postulated to be an aggravator of cardiac myocyte damage and necrosis. This theory is supported by multiple pieces of evidence, including the presence of inflammatory infiltrates in more than two-thirds of ACM hearts, detection of different cardiotropic viruses in sporadic cases of ACM, the fact that patients with ACM often fulfill the histological criteria of active myocarditis, and the abundance of anti–desmoglein-2, antiheart, and anti-intercalated disk autoantibodies in patients with arrhythmogenic right ventricular cardiomyopathy. In keeping with the frequent familial occurrence of ACM, it has been proposed that, in addition to genetic predisposition to progressive myocardial damage, a heritable susceptibility to viral infections and immune reactions may explain familial clustering of ACM. Moreover, considerable in vitro and in vivo evidence implicates activated inflammatory signaling in ACM. Although the role of inflammation/immune response in ACM is not entirely clear, inflammation as a driver of phenotype and a potential target for mechanism-based therapy warrants further research. This review discusses the present evidence supporting the role of inflammatory and immune responses in ACM pathogenesis and proposes opportunities for translational and clinical investigation.

Naxos disease is a recessively inherited pattern of arrhythmogenic cardiomyopathy with palmoplantar keratoderma and woolly hair. The causative mutation identified in plakoglobin protein gene indicated a potential role of the desmosomal protein complex as culprit for cardiomyopathy. In the context of a family, the early evident cutaneous features may serve as a clinical screening tool to spot arrhythmogenic cardiomyopathy in subclinical stage. "Myocarditis-like episodes" may step up the disease evolution or mark a transition from concealed to symptomatic cardiomyopathy phase. Arrhythmogenic cardiomyopathy in Naxos disease shows increased penetrance and phenotypic expression but its arrhythmic risk is analogous to dominant forms.

BACKGROUND: Electrophysiological, imaging, and pathological studies have reported the presence of subtle structural abnormalities in hearts from patients with Brugada syndrome (BrS). However, data concerning disease involvement outside of the right ventricular outflow tract are limited. OBJECTIVES: This study sought to characterize the presence and distribution of ventricular myocardial fibrosis in a cohort of decedents experiencing sudden cardiac death caused by BrS. METHODS: The authors evaluated 28 whole hearts from consecutive sudden cardiac death cases attributed to BrS and 29 hearts from a comparator group comprised of noncardiac deaths (control subjects). Cardiac tissue from 6 regions across the right and left ventricle were stained with Picrosirius red for collagen and tissue composition was determined using image analysis software. Postmortem genetic testing was performed in cases with DNA retained for analysis. RESULTS: Of 28 BrS decedents (75% men; median age of death 25 years), death occurred in sleep or at rest in 24 of 28 (86%). The highest proportion of collagen was observed in the epicardial right ventricular outflow tract of the BrS group (23.7%; 95% CI: 20.8%-26.9%). Ventricular myocardium from BrS decedents demonstrated a higher proportion of collagen compared with control subjects (ratio 1.45; 95% CI: 1.22-1.71; P < 0.001), with no significant interactions with respect to sampling location or tissue layer. There was insufficient evidence to support differences in collagen proportion in SCN5A-positive cases (n = 5) when compared with control subjects (ratio 1.23; 95% CI: 0.75-1.43; P = 0.27). CONCLUSIONS: Brugada syndrome is associated with increased collagen content throughout right and left ventricular myocardium, irrespective of sampling location or myocardial layer.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human cardiac disease. This pathway may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication. Genetic mutations in desmosomal genes resulting in the destabilization/breakdown of the desmosomal proteome are a central hallmark of all genetic-based desmosomal-targeted diseases, including the cardiac disease arrhythmogenic right ventricular (RV) dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here we identified a desmosomal resident regulatory complex, composed of subunit 6 of the COP9 signalosome (CSN6), enzymatically restricted neddylation and targets desmosomal proteome. Pharmacological restoration of CSN enzymatic function (via neddylation inhibitors) could rescue desmosomal protein loss in CSN6 deficient cardiomyocytes. Through the generation of two novel mouse models, we showed that cardiomyocyte-restricted CSN6 loss in mice selectively accelerated desmosomal destruction to trigger classic disease features associated with ARVD/C. We further showed that disruption of CSN6-mediated (neddylation) pathways underlined ARVD/C as CSN6 binding, localization, levels and function were impacted in hearts of classic ARVD/C mouse models and ARVD/C patients impacted by desmosomal loss and mutations, respectively. We anticipate our findings have broad implications towards understanding mechanisms driving desmosome degradation in other desmosomal-based diseases, such as cancers.

The diagnosis of arrhythmogenic cardiomyopathy (ACM) is challenging especially in children at risk of adverse events. Analysis of cardiac myocyte junctional protein distribution may have diagnostic and prognostic implications, but its utility is limited by the need for a myocardial sample. We previously reported that buccal mucosa cells show junctional protein redistribution similar to that seen in cardiac myocytes of adult patients with ACM.We aimed to determine when junctional protein distribution abnormalities first occur in children with ACM variants and whether they correlate with progression of clinically apparent disease.We analyzed buccal mucosa samples of children and adolescents with a family history of ACM (Junctional protein re-localization in buccal mucosa cells did not correlate with the presence of ACM-causing variants but instead occurred with clinical onset of disease. No changes in protein distribution were seen unless and until there was clinical evidence of disease. In addition, progressive shifts in the distribution of key proteins correlated with worsening of the disease phenotype. Finally, we observed restoration of junctional signal for Cx43 in patient with a favorable response to anti-arrhythmic therapy.Due to ethical concerns about obtaining heart biopsies in children with no apparent disease, it has not been possible to analyze molecular changes in cardiac myocytes with the onset/progression of clinical disease. Using buccal smears as a surrogate for the myocardium may facilitate future studies of mechanisms and pathophysiological consequences of junctional protein redistribution in ACM. Buccal cells may also be a safe and inexpensive tool for risk stratification and potentially monitoring response to treatment in children bearing ACM variants.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

Background: Inflammation is a prominent feature of arrhythmogenic cardiomyopathy (ACM), but whether it contributes to the disease phenotype is not known. Methods: To define the role of inflammation in the pathogenesis of ACM, we characterized nuclear factor-κB signaling in ACM models in vitro and in vivo and in cardiac myocytes from patient induced pluripotent stem cells. Results: Activation of nuclear factor-κB signaling, indicated by increased expression and nuclear accumulation of phospho-RelA/p65, occurred in both an in vitro model of ACM (expression of JUP 2157del2 in neonatal rat ventricular myocytes) and a robust murine model of ACM (homozygous knock-in of mutant desmoglein-2 [ Dsg2 mut/mu t ]) that recapitulates the cardiac manifestations seen in patients with ACM. Bay 11-7082, a small-molecule inhibitor of nuclear factor-κB signaling, prevented the development of ACM disease features in vitro (abnormal redistribution of intercalated disk proteins, myocyte apoptosis, release of inflammatory cytokines) and in vivo (myocardial necrosis and fibrosis, left ventricular contractile dysfunction, electrocardiographic abnormalities). Hearts of Dsg2 mut/mut mice expressed markedly increased levels of inflammatory cytokines and chemotactic molecules that were attenuated by Bay 11-7082. Salutary effects of Bay 11-7082 correlated with the extent to which production of selected cytokines had been blocked. Nuclear factor-κB signaling was also activated in cardiac myocytes derived from a patient with ACM. These cells produced and secreted abundant inflammatory cytokines under basal conditions, and this was also greatly reduced by Bay 11-7082. Conclusions: Inflammatory signaling is activated in ACM and drives key features of the disease. Targeting inflammatory pathways may be an effective new mechanism-based therapy for ACM.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2(mut/mut)), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca(2+)) overload in Dsg2(mut/mut) hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2(mut/mut) mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca(2+) overload–induced necrosis. When cardiomyocytes differentiated from Dsg2(mut/mut) embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2(mut/mut) ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Introduction Sarcoidosis is a potentially life-threatening, inflammatory, granulomatous disease that affects multiple organs including the heart. Heretofore, its unknown etiology had hindered the creation of experimental models and the understanding of the molecular mechanisms of pathogenesis behind it. Purpose To extensively phenotype the heart of the first mouse model of sarcoidosis created through deletion of the tuberous sclerosis 2 (Tsc2) gene in the CD11c-positive macrophage population. Methods Tsc2 fl/fl CD11c Cre+ (Tsc2-KO; n=7) and Tsc2 fl/fl CD11c Cre- (Tsc2-WT; n=7) mice were subjected to echocardiography at 25 weeks of age (woa) to assess myocardial dimensions and function. Hearts of 13 and 25woa animals were subjected to histological and immunological stains to assess tissue changes, subtype inflammatory infiltrates and examine the localization of key proteins shown to be re-distributed in patients. Results At 13 woa, Tsc2-KO animals show inflammatory infiltrates; subtyped mainly as macrophages as well as evidence of myocyte destruction. At 25 woa, the number of inflammatory cells is significantly higher and there is heavy fibrotic replacement primarily in the septum and trabeculae. Older animals also show giant cells and non-necrotizing granulomas. The hearts show heterogeneous gap junction remodeling known to constitute an arrhythmogenic substrate and lack of immunoreactive signal for the desmosomal protein plakoglobin from the cell-cell junctions just as described in patients. The left ventricular ejection fraction and LV morphology was not significantly different between the two groups (EF: 64±4% in Tsc2-KO vs 64±2% in Tsc2-WT; LV end-systolic diameter: 4.51±0.54 mm in Tsc2-KO vs 4.59±0.29 mm in Tsc2-WT). However, there was a strong trend towards increasing filling pressure (E/e'ratio; 14.24±4.01 vs 12.15±2.54) and mean pulmonary pressure (21±6 vs 18±3 mmHg) in Tsc2-KO mice compared to controls suggesting diastolic dysfunction. Conclusion Hearts of the Tsc2 fl/fl CD11c Cre+ animals show a phenotype highly reminiscent of cardiac sarcoidosis in patients. We anticipate that this model will be very useful in deciphering molecular mechanisms of pathogenesis as well as testing much-needed mechanism-based therapies. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation - PG/18/27/33616

BACKGROUND: Arrhythmogenic Cardiomyopathy (AC) is a heritable myocardial disorder and a major cause of sudden cardiac death. It is typically caused by mutations in desmosomal genes. Desmin gene (DES) variants have been previously reported in AC, but with insufficient evidence to support their pathogenicity.; METHODS: We aimed to assess a large AC patient cohort for DES mutations and describe a unique phenotype associated with a recurring variant in three families. A cohort of 138 probands with a diagnosis of AC and no identifiable desmosomal gene mutation were prospectively screened by whole exome sequencing.; RESULTS: A single DES variant (p.Leu115Ile, c.343C>A) was identified in three index patients (2%). We assessed the clinical phenotypes within their families and confirmed co-segregation. One carrier required heart transplantation, two died suddenly and one died of non-cardiac causes. All cases had right and left ventricular (LV) involvement. LV late gadolinium enhancement was present in all and circumferential sub-epicardial distribution was confirmed on histology. A significant burden of ventricular arrhythmias was noted. Desmin aggregates were not observed macroscopically but analysis of the desmin filament formation in transfected cardiomyocytes derived from induced pluripotent stem cells and SW13 cells revealed cytoplasmic aggregation of mutant desmin. Atomic force microscopy revealed that the mutant form accumulates into short proto-filaments and small fibrous aggregates.; CONCLUSIONS: DES p.Leu115Ile leads to disruption of the desmin filament network and causes a malignant biventricular form of AC, characterized by LV dysfunction and a circumferential subepicardial distribution of myocardial fibrosis. Copyright © 2020. Published by Elsevier Inc.

Background: Postoperative atrial fibrillation (POAF) occurs in 30% to 50% of patients undergoing cardiac surgery and is associated with increased morbidity and mortality. Prospective identification of structural/molecular changes in atrial myocardium that correlate with myocardial injury and precede and predict risk of POAF may identify new molecular pathways and targets for prevention of this common morbid complication. Methods: Right atrial appendage samples were prospectively collected during cardiac surgery from 239 patients enrolled in the OPERA trial (Omega-3 Fatty Acids for Prevention of Post-Operative Atrial Fibrillation), fixed in 10% buffered formalin, and embedded in paraffin for histology. We assessed general tissue morphology, cardiomyocyte diameters, myocytolysis (perinuclear myofibril loss), accumulation of perinuclear glycogen, interstitial fibrosis, and myocardial gap junction distribution. We also assayed NT-proBNP (N-terminal pro-B-type natriuretic peptide), hs-cTnT, CRP (C-reactive protein), and circulating oxidative stress biomarkers (F2-isoprostanes, F3-isoprostanes, isofurans) in plasma collected before, during, and 48 hours after surgery. POAF was defined as occurrence of postcardiac surgery atrial fibrillation or flutter of at least 30 seconds duration confirmed by rhythm strip or 12-lead ECG. The follow-up period for all arrhythmias was from surgery until hospital discharge or postoperative day 10. Results: Thirty-five percent of patients experienced POAF. Compared with the non-POAF group, they were slightly older and more likely to have chronic obstructive pulmonary disease or heart failure. They also had a higher European System for Cardiac Operative Risk Evaluation and more often underwent valve surgery. No differences in left atrial size were observed between patients with POAF and patients without POAF. The extent of atrial interstitial fibrosis, cardiomyocyte myocytolysis, cardiomyocyte diameter, glycogen score or Cx43 distribution at the time of surgery was not significantly associated with incidence of POAF. None of these histopathologic abnormalities were correlated with levels of NT-proBNP, hs-cTnT, CRP, or oxidative stress biomarkers. Conclusions: In sinus rhythm patients undergoing cardiac surgery, histopathologic changes in the right atrial appendage do not predict POAF. They also do not correlate with biomarkers of cardiac function, inflammation, and oxidative stress. Graphic Abstract: A graphic abstract is available for this article.

Background: Pathogenic variants in the filamin C (FLNC) gene are associated with inherited cardiomyopathies including dilated cardiomyopathy with an arrhythmogenic phenotype. We evaluated FLNC variants in arrhythmogenic cardiomyopathy (ACM) and investigated the disease mechanism at a molecular level. Methods: 120 gene-elusive ACM patients who fulfilled diagnostic criteria for arrhythmogenic right ventricular cardiomyopathy (ARVC) were screened by whole exome sequencing. Fixed cardiac tissue from FLNC variant carriers who had died suddenly was investigated by histology and immunohistochemistry. Results: Novel or rare FLNC variants, four null and five variants of unknown significance, were identified in nine ACM probands (7.5%). In FLNC null variant carriers (including family members, n = 16) Task Force diagnostic electrocardiogram repolarization/depolarization abnormalities were uncommon (19%), echocardiography was normal in 69%, while 56% had >500 ventricular ectopics/24 h or ventricular tachycardia on Holter and 67% had late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMRI). Ten gene positive individuals (63%) had abnormalities on ECG or CMRI that are not included in the current diagnostic criteria for ARVC. Immunohistochemistry showed altered key protein distribution, distinctive from that observed in ARVC, predominantly in the left ventricle. Conclusions: ACM associated with FLNC variants presents with a distinctive phenotype characterized by Holter arrhythmia and LGE on CMRI with unremarkable ECG and echocardiographic findings. Clinical presentation in asymptomatic mutation carriers at risk of sudden death may include abnormalities which are currently non-diagnostic for ARVC. At the molecular level, the pathogenic mechanism related to FLNC appears different to classic forms of ARVC caused by desmosomal mutations. Keywords: ARVC; Arrhythmogenic cardiomyopathy; Filamin C variants; Immunohistochemistry; Late gadolinium enhancement.

Arrhythmogenic cardiomyopathy (ACM) encompasses a group of inherited cardiomyopathies including arrhythmogenic right ventricular cardiomyopathy (ARVC) whose molecular disease mechanism is associated with dysregulation of the canonical WNT signalling pathway. Recent evidence indicates that ARVC and ACM caused by pathogenic variants in the FLNC gene encoding filamin C, a major cardiac structural protein, may have different molecular mechanisms of pathogenesis. We sought to identify dysregulated biological pathways in FLNC-associated ACM. RNA was extracted from seven paraffin-embedded left ventricular tissue samples from deceased ACM patients carrying FLNC variants and sequenced. Transcript levels of 623 genes were upregulated and 486 genes were reduced in ACM in comparison to control samples. The cell adhesion pathway and ILK signalling were among the prominent dysregulated pathways in ACM. Consistent with these findings, transcript levels of cell adhesion genes JAM2, NEO1, VCAM1 and PTPRC were upregulated in ACM samples. Moreover, several actin-associated genes, including FLNC, VCL, PARVB and MYL7, were suppressed, suggesting dysregulation of the actin cytoskeleton. Analysis of the transcriptome for dysregulated biological pathways predicted activation of inflammation and apoptosis and suppression of oxidative phosphorylation and MTORC1 signalling in ACM. Our data suggests dysregulated cell adhesion and ILK signalling as novel putative pathogenic mechanisms of ACM caused by FLNC variants which are distinct from the postulated disease mechanism of classic ARVC caused by desmosomal gene mutations. This knowledge could help in the design of future gene therapy strategies which would target specific components of these pathways and potentially lead to novel treatments for ACM.

The original designation of "Arrhythmogenic right ventricular (dysplasia/) cardiomyopathy"(ARVC) was used by the scientists who first discovered the disease, in the pre-genetic and pre-cardiac magnetic resonance era, to describe a new heart muscle disease predominantly affecting the right ventricle, whose cardinal clinical manifestation was the occurrence of malignant ventricular arrhythmias. Subsequently, autopsy investigations, genotype-phenotype correlations studies and the increasing use of contrast-enhancement cardiac magnetic resonance showed that the fibro-fatty replacement of the myocardium represents the distinctive phenotypic feature of the disease that affects the myocardium of both ventricles, with left ventricular involvement which may parallel or exceed the severity of right ventricular involvement. This has led to the new designation of "Arrhythmogenic Cardiomyopathy" (ACM), that represents the evolution of the original term of ARVC. The present International Expert Consensus document proposes an upgrade of the criteria for diagnosis of the entire spectrum of the phenotypic variants of ACM. The proposed "Padua criteria" derive from the diagnostic approach to ACM, which has been developed over 30 years by the multidisciplinary team of basic researchers and clinical cardiologists of the Medical School of the University of Padua. The Padua criteria are a working framework to improve the diagnosis of ACM by introducing new diagnostic criteria regarding tissue characterization findings by contrast-enhanced cardiac magnetic resonance, depolarization/repolarization ECG abnormalities and ventricular arrhythmia features for diagnosis of the left ventricular phenotype. The proposed diagnostic criteria need to be further validated by future clinical studies in large cohorts of patients.

Objectives The purpose of this study was to assess the phenotype of Filamin C (FLNC) truncating variants in dilated cardiomyopathy (DCM) and understand the mechanism leading to an arrhythmogenic phenotype. Background Mutations in FLNC are known to lead to skeletal myopathies, which may have an associated cardiac component. Recently, the clinical spectrum of FLNC mutations has been recognized to include a cardiac-restricted presentation in the absence of skeletal muscle involvement. Methods A population of 319 U.S. and European DCM cardiomyopathy families was evaluated using whole-exome and targeted next-generation sequencing. FLNC truncation probands were identified and evaluated by clinical examination, histology, transmission electron microscopy, and immunohistochemistry. Results A total of 13 individuals in 7 families (2.2%) were found to harbor 6 different FLNC truncation variants (2 stopgain, 1 frameshift, and 3 splicing). Of the 13 FLNC truncation carriers, 11 (85%) had either ventricular arrhythmias or sudden cardiac death, and 5 (38%) presented with evidence of right ventricular dilation. Pathology analysis of 2 explanted hearts from affected FLNC truncation carriers showed interstitial fibrosis in the right ventricle and epicardial fibrofatty infiltration in the left ventricle. Ultrastructural findings included occasional disarray of Z-discs within the sarcomere. Immunohistochemistry showed normal plakoglobin signal at cell–cell junctions, but decreased signals for desmoplakin and synapse-associated protein 97 in the myocardium and buccal mucosa. Conclusions We found FLNC truncating variants, present in 2.2% of DCM families, to be associated with a cardiac-restricted arrhythmogenic DCM phenotype characterized by a high risk of life-threatening ventricular arrhythmias and a pathological cellular phenotype partially overlapping with arrhythmogenic right ventricular cardiomyopathy.

Mechanisms underlying the development of idiopathic dilated cardiomyopathy (DCM) remain poorly understood. Using transcription factor expression profiling, we identified estrogen-related receptor-β (ESRRβ), a member of the nuclear receptor family of transcription factors, as highly expressed in murine hearts and other highly oxidative striated muscle beds. Mice bearing cardiac-specific deletion of ESRRβ (MHC-ERRB KO) develop DCM and sudden death at ~10 mo of age. Isolated adult cardiomyocytes from the MHC-ERRB KO mice showed an increase in calcium sensitivity and impaired cardiomyocyte contractility, which preceded echocardiographic cardiac remodeling and dysfunction by several months. Histological analyses of myocardial biopsies from patients with various cardiomyopathies revealed that ESRRβ protein is absent from the nucleus of cardiomyocytes from patients with DCM but not other forms of cardiomyopathy (ischemic, hypertrophic, and arrhythmogenic right ventricular cardiomyopathy). Taken together these observations suggest that ESRRβ is a critical component in the onset of DCM by affecting contractility and calcium balance. NEW & NOTEWORTHY Estrogen-related receptor-β (ESRRβ) is highly expressed in the heart and cardiac-specific deletion results in the development of a dilated cardiomyopathy (DCM). ESRRβ is mislocalized in human myocardium samples with DCM, suggesting a possible role for ESRRβ in the pathogenesis of DCM in humans.

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Introduction Arrhythmogenic Cardiomyopathy (AC) is typically caused by mutations in the desmosomal genes, however non-desmosomal genes have been increasingly implicated. Desmin gene (DES) mutations have been previously reported in AC, but in many cases there are insufficient data to support their pathogenicity. Purpose We assessed our AC cohort for DES gene mutations and describe the clinical phenotype associated with a recurring variant present in 3 unrelated families. Methods Genetic testing was performed using next-generation sequencing for 41 genes in a total of 138 AC probands with a definite diagnosis of AC based on the revised 2010 Task Force diagnostic criteria. All candidate variants were confirmed using Sanger sequencing. Clinical and genetic cascade screening were expanded to the first-degree relatives of the probands. Retained tissue from deceased individuals was used for genetic testing. All living mutation carriers underwent clinical assessment including physical examination, 12-lead ECG, signal-averaged ECG, echocardiography, cardiac magnetic resonance imaging (MRI) and 24h Holter-monitoring. Results Two DES gene variants, p.Ser298Leu (n=1) and p.Leu115Ile (n=3), were identified in 4 out of the 138 probands (3%). The former coexisted with a pathogenic DSP gene mutation and has not been further evaluated. The latter is a novel variant, absent in control databases (gnomAD) and was the only variant present in 3 unrelated families (see figure). One carrier required heart transplant (A-II-1), two died suddenly (A-III-1, B-II-1) and one died of non-cardiac causes (B-I-2). Detailed clinical information was present in 8 mutation carriers (2 male, age 45±19 years). Seven (88%) had a definite diagnosis and one had a borderline diagnosis of AC. All cases (100%) had right ventricular (RV) wall motion abnormalities, 6 (75%) had a dilated RV, 6 (75%) a dilated LV and 6 (75%) had LV dysfunction (mild in 5 and severe in 1). LV late gadolinium enhancement (LGE) was present in all 6 carriers that had a cardiac MRI with a circumferential sub-epicardial distribution (see figure, case A-III-2). Non-sustained ventricular tachycardia (VT) was present in 7 (88%) and sustained VT in 2 cases (25%). The ventricular ectopic burden per 24h ranged from 426 to 10583 with a median value of 820. Figure 1 Conclusion Variants of the DES gene are rare causes of AC. The novel p.Leu115Ile variant seems to be prevalent in a large UK-based cohort and it causes a biventricular form of AC, with a characteristic scar pattern on MRI and severe outcomes. Acknowledgement/Funding Alexandros Protonotarios is supported by a BHF Clinical Research Training Fellowship no. FS/18/82/34024

It is 35 years since the first description of arrhythmogenic right ventricular cardiomyopathy (ARVC) and more than 20 years since the first reports establishing desmosomal gene mutations as a major cause of the disease. Early advances in the understanding of the clinical, pathological and genetic architecture of ARVC resulted in consensus diagnostic criteria, which proved to be sensitive but not entirely specific for the disease. In more recent years, clinical and genetic data from families and the recognition of a much broader spectrum of structural disorders affecting both ventricles and associated with a propensity to ventricular arrhythmia have raised many questions about pathogenesis, disease terminology and clinical management. In this paper, we present the conclusions of an expert round table that aimed to summarise the current state of the art in arrhythmogenic cardiomyopathies and to define future research priorities.

Phospholamban (PLN) p.Arg14del cardiomyopathy is characterized by a distinct arrhythmogenic biventricular phenotype that can be predominantly left ventricular, right ventricular, or both. Our aim was to further elucidate distinct features of this cardiomyopathy with respect to the distribution of desmosomal proteins observed by immunofluorescence (IF) in comparison to desmosomal arrhythmogenic cardiomyopathy and co-existent genetic variants. We studied eight explanted heart specimens from PLN p.Arg14del mutation carriers. Macro- and microscopic examination revealed biventricular presence of fibrofatty replacement and interstitial fibrosis. Five out of 8 (63%) patients met consensus criteria for both arrhythmogenic right ventricular cardiomyopathy (ARVC) and dilated cardiomyopathy (DCM). In four cases, targeted next-generation sequencing revealed one additional pathogenic variant and six variants of unknown significance. IF showed diminished junction plakoglobin signal intensity at the intercalated disks in 4 (67%) out of 6 cases fulfilling ARVC criteria but normal intensity in both cases fulfilling only DCM criteria. Notably, the four cases with diminished junction plakoglobin were also those where an additional gene variant was detected. IF for two proteins recently investigated in desmosomal arrhythmogenic cardiomyopathy (ACM), synapse-associated protein 97 and glycogen synthase kinase-3 beta, showed a distinct distributional pattern in comparison to desmosomal ACM. In 7 (88%) out of 8 cases we observed both a strong synapse-associated protein 97 signal at the sarcomeres and no glycogen synthase kinase-3 beta translocation to the intercalated discs. Phospholamban p.Arg14del cardiomyopathy is characterized by a distinct molecular signature compared to desmosomal ACM, specifically a different desmosomal protein distribution. This study substantiates the idea that additional genetic variants play a role in the phenotypical heterogeneity. (C) 2018 Elsevier Inc. All rights reserved.

BACKGROUND Cardiomyopathies are usually inherited and predominantly affect adults, but they can also present in childhood. Although our understanding of the molecular basis of pediatric cardiomyopathy has improved, the underlying mechanism remains elusive in a substantial proportion of cases.OBJECTIVES This study aimed to identify new genes involved in pediatric cardiomyopathy.METHODS The authors performed homozygosity mapping and whole-exome sequencing in 2 consanguineous families with idiopathic pediatric cardiomyopathy. Sixty unrelated patients with pediatric cardiomyopathy were subsequently screened for mutations in a candidate gene. First-degree relatives were submitted to cardiac screening and cascade genetic testing. Myocardial samples from 2 patients were processed for histological and immunohistochemical studies.RESULTS We identified 5 patients from 3 unrelated families with pediatric cardiomyopathy caused by homozygous truncating mutations in ALPK3, a gene encoding a nuclear kinase that plays an essential role in early differentiation of cardiomyocytes. All patients with biallelic mutations presented with severe hypertrophic and/or dilated cardiomyopathy in utero, at birth, or in early childhood. Three patients died from heart failure within the first week of life. Moreover, 2 of 10 (20%) heterozygous family members showed hypertrophic cardiomyopathy with an atypical distribution of hypertrophy. Deficiency of alpha-kinase 3 has previously been associated with features of both hypertrophic and dilated cardiomyopathy in mice. Consistent with studies in knockout mice, we provide microscopic evidence for intercalated disc remodeling.CONCLUSIONS Biallelic truncating mutations in the newly identified gene ALPK3 give rise to severe, early-onset cardiomyopathy in humans. Our findings highlight the importance of transcription factor pathways in the molecular mechanisms underlying human cardiomyopathies. (C) 2016 by the American College of Cardiology Foundation.

We read with interest the provocative work by Pieroni et al. [(1)][1] describing the relationship between electroanatomic abnormalities and pathological substrate in Brugada syndrome (BrS). In particular, we noted that right ventricular (RV) biopsy guided by 3-dimensional RV electroanatomic mapping

Arrhythmogenic cardiomyopathy (ACM) is a primary myocardial disease. It is characterized by frequent ventricular arrhythmias and increased risk of sudden cardiac death typically arising as an early manifestation before the onset of significant myocardial remodelling. Myocardial degeneration, often confined to the right ventricular free wall, with replacement by fibrofatty scar tissue, develops in many patients. ACM is a familial disease but genetic penetrance can be low and disease expression is highly variable. Inflammation might promote disease progression. It also appears that exercise increases disease penetrance and accelerates its development. More than 60% of probands harbour mutations in genes that encode desmosomal proteins, which has raised the possibility that defective cell-cell adhesion might play a role in disease pathogenesis. Recent advances have implicated changes in the canonical wingless-type mouse mammary tumour virus integration site (Wnt)/β-catenin and Hippo signalling pathways and defects in forwarding trafficking of ion channels and other proteins to the intercalated disk in cardiac myocytes. In this review we summarize the current understanding of the pathogenesis of ACM and highlight future research directions.

Objective Cathepsin K and tartrate-resistant acid phosphatase (TRAP) are two proteins expressed in osteoclastic giant cells. Recently we showed that lesional multinucleated giant cells (MNGs) in pulmonary granulomatosis with polyangiitis expressed these proteins. We aimed to clarify whether the expression of these two proteins has any specificity or is a general feature of MNGs associated with multiple types of granulomatous inflammation. Methods In total, 7 Crohn's disease (CD), 5 GCA, 5 giant cell myocarditis (GCM), 11 sarcoidosis and 6 tuberculosis cases were examined for expression of cathepsin K and TRAP using immunohistochemistry (IHC). Protein expression was semi-quantitatively classified as none, weak, moderate or strong. In addition, tissue TRAP activity was examined using an enzymatic reaction. Results The expression of cathepsin K was robust in >95% of MNGs of all examined disease groups, whereas TRAP expression varied; CD, GCA and tuberculosis showed strong TRAP expression. TRAP expression in sarcoidosis and GCM was weaker (CD vs GCM, P = 0.04; CD vs sarcoidosis, P = 0.06). Compared with IHC, TRAP detection using an enzymatic colour reaction had limited sensitivity. Conclusion Expression of TRAP and cathepsin K is a general feature of MNGs and their expression might be related to histopathological pattern.

Arrhythmogenic cardiomyopathy (ACM) is characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. We previously reported that SB216763 (SB2), annotated as a GSK3β inhibitor, reverses disease phenotypes in a zebrafish model of ACM. Here, we show that SB2 prevents myocyte injury and cardiac dysfunction in vivo in two murine models of ACM at baseline and in response to exercise. SB2-treated mice with desmosome mutations showed improvements in ventricular ectopy and myocardial fibrosis/inflammation as compared with vehicle-treated (Veh-treated) mice. GSK3β inhibition improved left ventricle function and survival in sedentary and exercised Dsg2mut/mut mice compared with Veh-treated Dsg2mut/mut mice and normalized intercalated disc (ID) protein distribution in both mutant mice. GSK3β showed diffuse cytoplasmic localization in control myocytes but ID redistribution in ACM mice. Identical GSK3β redistribution is present in ACM patient myocardium but not in normal hearts or other cardiomyopathies. SB2 reduced total GSK3β protein levels but not phosphorylated Ser 9–GSK3β in ACM mice. Constitutively active GSK3β worsens ACM in mutant mice, while GSK3β shRNA silencing in ACM cardiomyocytes prevents abnormal ID protein distribution. These results highlight a central role for GSKβ in the complex phenotype of ACM and provide further evidence that pharmacologic GSKβ inhibition improves cardiomyopathies due to desmosome mutations.

Background— Analysis of myocardium has revealed mechanistic insights into arrhythmogenic cardiomyopathy but cardiac samples are difficult to obtain from probands and especially from family members. To identify a potential surrogate tissue, we characterized buccal mucosa cells. Methods and Results— Buccal cells from patients, mutation carriers, and controls were immunostained and analyzed in a blinded fashion. In additional studies, buccal cells were grown in vitro and incubated with SB216763. Immunoreactive signals for the desmosomal protein plakoglobin and the major cardiac gap junction protein Cx43 were markedly diminished in buccal mucosa cells from arrhythmogenic cardiomyopathy patients with known desmosomal mutations when compared with controls. Plakoglobin and Cx43 signals were also reduced in most family members who carried disease alleles but showed no evidence of heart disease. Signal for the desmosomal protein plakophilin-1 was reduced in buccal mucosa cells in patients with PKP2 mutations but not in those with mutations in other desmosomal genes. Signal for the desmosomal protein desmoplakin was reduced in buccal mucosa cells from patients with mutations in DSP , DSG2 , or DSC2 but not in PKP2 or JUP . Abnormal protein distributions were reversed in cultured cells incubated with SB216763, a small molecule that rescues the disease phenotype in cardiac myocytes. Conclusions— Buccal mucosa cells from arrhythmogenic cardiomyopathy patients exhibit changes in the distribution of cell junction proteins similar to those seen in the heart. These cells may prove useful in future studies of disease mechanisms and drug screens for effective therapies in arrhythmogenic cardiomyopathy.

Aims Anecdotal observations suggest that sub-clinical electrophysiological manifestations of arrhythmogenic right ventricular cardiomyopathy (ARVC) develop before detectable structural changes ensue on cardiac imaging. To test this hypothesis, we investigated a murine model with conditional cardiac genetic deletion of one desmoplakin allele (DSP ±) and compared the findings to patients with non-diagnostic features of ARVC who carried mutations in desmoplakin. Methods and results Murine: the DSP (±) mice underwent electrophysiological, echocardiographic, and immunohistochemical studies. They had normal echocardiograms but delayed conduction and inducible ventricular tachycardia associated with mislocalization and reduced intercalated disc expression of Cx43. Sodium current density and myocardial histology were normal at 2 months of age. Human: ten patients with heterozygous mutations in DSP without overt structural heart disease (DSP+) and 12 controls with supraventricular tachycardia were studied by high-density electrophysiological mapping of the right ventricle. Using a standard S1–S2 protocol, restitution curves of local conduction and repolarization parameters were constructed. Significantly greater mean increases in delay were identified particularly in the outflow tract vs. controls (P< 0.01) coupled with more uniform wavefront progression. The odds of a segment with a maximal activation–repolarization interval restitution slope >1 was 99% higher (95% CI: 13%; 351%, P= 0.017) in DSP+ vs. controls. Immunostaining revealed Cx43 mislocalization and variable Na channel distribution. Conclusion Desmoplakin disease causes connexin mislocalization in the mouse and man preceding any overt histological abnormalities resulting in significant alterations in conduction–repolarization kinetics prior to morphological changes detectable on conventional cardiac imaging. Haploinsufficiency of desmoplakin is sufficient to cause significant Cx43 mislocalization. Changes in sodium current density and histological abnormalities may contribute to a worsening phenotype or disease but are not necessary to generate an arrhythmogenic substrate. This has important implications for the earlier diagnosis of ARVC and risk stratification.

We present the case of a patient who underwent cardiac transplantation with the diagnosis of idiopathic dilated cardiomyopathy. Once the explanted heart was examined, a type of granulomatous myocarditis compatible with cardiac sarcoidosis was observed. However, there was severe involvement of the right ventricle, with markedly reduced width of the muscular layer and extensive fibrofatty replacement, findings similar to the ones encountered in cases of arrhythmogenic right ventricular cardiomyopathy (ARVC). Confocal immunofluorescence analysis revealed a reduced signal for plakoglobin and desmoplakin at the cardiac intercalated disks. The immunoreactive signal for desmin showed the typical sarcomeric distribution but not a concentrated signal at the intercalated disks, a pattern previously seen in an 11-year-old girl with Carvajal syndrome bearing a C-terminal truncating mutation in the desmoplakin gene. This case illustrates the difficult and challenging work involved in performing a differential diagnosis among idiopathic dilated cardiomyopathy, isolated cardiac sarcoidosis, and ARVC, all of which are clinical entities known to masquerade as one another.

Background— Immunoreactive signal for the desmosomal protein plakoglobin (γ-catenin) is reduced at cardiac intercalated disks in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a highly arrhythmogenic condition caused by mutations in genes encoding desmosomal proteins. Previously, we observed a false-positive case in which plakoglobin signal was reduced in a patient initially believed to have ARVC but who actually had cardiac sarcoidosis. Sarcoidosis can masquerade clinically as ARVC but has not been previously associated with altered desmosomal proteins. Methods and Results— We observed marked reduction in immunoreactive signal for plakoglobin at cardiac myocyte junctions in patients with sarcoidosis and giant cell myocarditis, both highly arrhythmogenic forms of myocarditis associated with granulomatous inflammation. In contrast, plakoglobin signal was not depressed in lymphocytic (nongranulomatous) myocarditis. To determine whether cytokines might promote dislocation of plakoglobin from desmosomes, we incubated cultures of neonatal rat ventricular myocytes with selected inflammatory mediators. Brief exposure to low concentrations of interleukin (IL)-17, tumor necrosis factor-α (TNF-α), and IL-6 (cytokines implicated in granulomatous myocarditis) caused translocation of plakoglobin from cell-cell junctions to intracellular sites, whereas other potent cytokines implicated in nongranulomatous myocarditis had no effect, even at much higher concentrations. We also observed myocardial expression of IL-17 and TNF-α and elevated levels of serum inflammatory mediators, including IL-6R, IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β, in patients with ARVC (all P Conclusions— The results suggest novel disease mechanisms involving desmosomal proteins in granulomatous myocarditis and implicate cytokines, perhaps derived in part from the myocardium, in disruption of desmosomal proteins and arrhythmogenesis in ARVC.

Left dominant arrhythmogenic cardiomyopathy (LDAC) exhibits characteristic phenotypic and genetic features which were found in the five Spanish family members described in this study. Triggered by a cold, a young man presented with a ventricular tachycardia of left ventricular origin and left ventricular late gadolinium enhancement. His resting ECG showed low potentials, delayed ventricular depolarization (inferior and V4-V6 leads) and atrioventricular conduction disturbances. His endomyocardial biopsy revealed myocyte loss with interstitial fibrosis. Despite the initial diagnosis of myocarditis, familial screening was pivotal in confirming the diagnosis of LDAC. A novel nonsense mutation in the desmoplakin gene (Q1866X) and the truncated protein which it produces were observed in skin samples.

Left dominant arrhythmogenic cardiomyopathy (LDAC) exhibits characteristic phenotypic and genetic features which were found in the five Spanish family members described in this study. Triggered by a cold, a young man presented with a ventricular tachycardia of left ventricular origin and left ventricular late gadolinium enhancement. His resting ECG showed low potentials, delayed ventricular depolarization (inferior and V4-V6 leads) and atrioventricular conduction disturbances. His endomyocardial biopsy revealed myocyte loss with interstitial fibrosis. Despite the initial diagnosis of myocarditis, familial screening was pivotal in confirming the diagnosis of LDAC. A novel nonsense mutation in the desmoplakin gene (Q1866X) and the truncated protein which it produces were observed in skin samples.

Background Cellular adhesion mediated by cardiac desmosomes is a prerequisite for proper electric propagation mediated by gap junctions in the myocardium. However, the molecular principles underlying this interdependence are not fully understood. Objective The purpose of this study was to determine potential causes of right ventricular conduction abnormalities in a patient with borderline diagnosis of arrhythmogenic right ventricular cardiomyopathy. Methods To assess molecular changes, the patient's myocardial tissue was analyzed for altered desmosomal and gap junction (connexin43) protein levels and localization. In vitro functional studies were performed to characterize the consequences of the desmosomal mutations. Results Loss of plakoglobin signal was evident at the cell junctions despite expression of the protein at control levels. Although the distribution of connexin43 was not altered, total protein levels were reduced and changes in phosphorylation were observed. The truncation mutant in desmocollin-2a is deficient in binding plakoglobin. Moreover, the ability of desmocollin-2a to directly interact with connexin43 was abolished by the mutation. No pathogenic potential of the desmoglein-2 missense change was identified. Conclusion The observed abnormalities in gap junction protein expression and phosphorylation, which precede an overt cardiac phenotype, likely are responsible for slow myocardial conduction in this patient. At the molecular level, altered binding properties of the desmocollin-2a mutant may contribute to the changes in connexin43. In particular, the newly identified interaction between the desmocollin-2a isoform and connexin43 provides novel insights into the molecular link between desmosomes and gap junctions.

Aims To evaluate arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) in affected families with desmosome mutations on the basis of the recently revised Task Force Criteria (TFC). Methods and results One hundred and three consecutive carriers of pathogenic desmosome mutations and 102 mutation-negative relatives belonging to 22 families with dominant and 14 families with recessive ARVC/D were evaluated according to the original and revised TFC. Serial cardiac assessment with 12-lead, signal-averaged, and 24 h ambulatory ECG and two-dimensional echocardiography was performed. Clinical events and outcome were prospectively analysed up to 24 years (median 4 years). With the revised criteria, 16 carriers were newly diagnosed on the basis of ECG abnormalities in 100%, ventricular arrhythmias in 79%, and functional/structural alterations in 31%, increasing diagnostic sensitivity from 57 to 71% ( P = 0.001). Task Force Criteria specificity improved from 92 to 99% ( P = 0.016). In dominant mutation carriers, penetrance changed significantly (61 vs. 42%, P = 0.001); no changes were observed in recessive homozygous carriers (97 vs. 97%, P = 1.00). Affected carriers according to the revised TFC ( n = 73) had 12-lead ECG abnormalities in 96%, ventricular arrhythmias in 91%, and functional/structural alterations fulfilling echocardiographic criteria in 76%. Cumulative and event-free survival did not differ significantly between dominant and recessive affected carriers, being at 78.6 vs. 76 and 51.7 vs. 55.4%, respectively, by the age of 40 years. Conclusion Revised TFC increased diagnostic sensitivity particularly in dominant ARVC/D. Serial family evaluation may rely on electrocardiography which seems to have the best diagnostic utility particularly in early disease that is not detectable by two-dimensional echocardiography.

The Role of Endomyocardial Biopsy in ARVC. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by a high incidence of ventricular tachyarrhythmias and sudden cardiac death that appear early in the natural history of the disease and may precede significant ventricular remodeling. The classical pathology of ARVC is degeneration of right ventricular free wall myocardium and its replacement by fat and fibrous tissue. The clinical presentation may be highly variable, however, and genetic penetrance is typically low which makes definitive diagnosis difficult, especially in early stages of the disease. Endomyocardial biopsy (EMB) has not been used widely in the diagnosis of ARVC, in part because pathological changes are usually most conspicuous in the epicardium of the right ventricular free wall and tend to spare the endocardium and interventricular septum. Thus, diagnostic pathological features of ARVC are often not seen in conventional septal biopsies. Diagnostic yield may be increased by sampling the RV free wall or by using 3-dimensional electroanatomic voltage mapping to identify affected areas, but these approaches can carry increased risk and require specialized equipment and experience. Moreover, diagnostic pathological changes may not be apparent in early disease despite an increased risk of arrhythmias and sudden death. This review considers the role of EMB in the diagnosis of ARVC and highlights recent advances in identifying potential tissue biomarkers that arise early in the disease process and occur diffusely throughout the myocardium. Analysis of conventional EMB using such biomarkers could improve diagnostic sensitivity and accuracy but widespread clinical application of this approach requires further validation.

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.

Background The diagnosis of arrhythmogenic right ventricular cardiomyopathy can be challenging. Disease-causing mutations in desmosomal genes have been identified. A novel diagnostic feature, loss of immunoreactivity for plakoglobin from the intercalated disks, recently was proposed. Objective The purpose of this study was to identify two novel mutations in the intracellular cadherin segment of desmoglein-2 (G812S and C813R in exon 15). Co-segregation of the G812S mutation with disease expression was established in a large Caucasian family. Endomyocardial biopsies of two individuals showed reduced plakoglobin signal at the intercalated disk. Methods To understand the pathologic changes occurring in the diseased myocardium, functional studies on three mutations in exon 15 of desmoglein-2 (G812C, G812S, C813R) were performed. Results Localization studies failed to detect any differences in targeting or stability of the mutant proteins, suggesting that they act via a dominant negative mechanism. Binding assays were performed to probe for altered binding affinities toward other desmosomal proteins, such as plakoglobin and plakophilin-2. Although no differences were observed for the mutated proteins in comparison to wild-type desmoglein-2, binding to plakophilin-2 depended on the expression system (i.e., bacterial vs mammalian protein expression). In addition, abnormal migration of the C813R mutant protein was observed in gel electrophoresis. Conclusion Loss of plakoglobin immunoreactivity from the intercalated disks appears to be the endpoint of complex pathologic changes, and our functional data suggest that yet unknown posttranslational modifications of desmoglein-2 might be involved.

BACKGROUND Desmin-related myopathy is a clinically heterogenous group of disorders encompassing myopathies, cardiomyopathies, conduction disease, and combinations of these disorders. Mutations in the gene encoding desmin (DES), a major intermediate filament protein, can underlie this phenotype.OBJECTIVE The purpose of this study was to investigate the clinical and pathologic characteristics of 27 patients from five families with an identical mutation in the head domain region (p.S13F) of desmin.METHODS/RESULTS ALL 27 carriers or obligate carriers of a p.S13F DES founder mutation demonstrated a fully penetrant yet variable phenotype. ALL patients demonstrated cardiac involvement characterized by high-grade AV block at young ages and important right ventricular (RV) involvement. RV predominance was demonstrated by the presence of right bundle branch block in 10 patients (sometimes as a first manifestation) and by RV heart failure in 6 patients, including 2 patients who fulfilled the diagnostic criteria for arrhythmogenic RV cardiomyopathy. Because of this clinical overlap with desmosome cardiomyopathies, we also studied the organization of the intercalated disks, particularly the distribution of desmosomal proteins. Normal amounts of the major desmosomal proteins were found, but the intercalated disks were more convoluted and elongated and had a zigzag appearance.CONCLUSION In this Largest series to date of individuals with a single head domain DES mutation, patients show a variable yet predominantly cardiologic phenotype characterized by conduction disease at an early age and RV involvement including right bundle branch block and/or RV tachycardias and arrhythmogenic RV cardiomyopathy phenocopies. A Localized effect of desmin on the structure of the cardiac intercalated disks might contribute to disease pathogenesis.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder associated with arrhythmias and sudden death. A recessive mutation in the gene encoding plakoglobin has been shown to cause Naxos disease, a cardiocutaneous syndrome characterized by ARVC and abnormalities of hair and skin. Here, we report, for the first time, a dominant mutation in the gene encoding plakoglobin in a German family with ARVC but no cutaneous abnormalities. The mutation (S39_K40insS) is predicted to insert an extra serine residue at position 39 in the N-terminus of plakoglobin. Analysis of a biopsy sample of the right ventricle from the proband showed markedly decreased localization of plakoglobin, desmoplakin, and connexin43 at intercalated discs in cardiac myocytes. A yeast-two-hybrid screen revealed that the mutant protein established novel interactions with histidine-rich calcium-binding protein and TGF beta induced apoptosis protein 2. Immunoblotting and confocal microscopy in human embryonic kidney 293 (HEK293) cell lines transfected to stably express either wild-type or mutant plakoglobin protein showed that the mutant protein was apparently ubiquitylated and was preferentially located in the cytoplasm, suggesting that the S39_K40insS mutation may increase plakoglobin turnover via proteasomal degradation. HEK293 cells expressing mutant plakoglobin also showed higher rates of proliferation and lower rates of apoptosis than did cells expressing the wild-type protein. Electron microscopy showed smaller and fewer desmosomes in cells expressing mutant plakoglobin. Taken together, these observations suggest that the S39_K40insS mutation affects the structure and distribution of mechanical and electrical cell junctions and could interfere with regulatory mechanisms mediated by Wnt-signaling pathways. These results implicate novel molecular mechanisms in the pathogenesis of ARVC.

Background— According to clinical-pathological correlation studies, the natural history of arrhythmogenic right ventricular dysplasia/cardiomyopathy is purported to progress from localized to global right ventricular dysfunction, followed by left ventricular (LV) involvement and biventricular pump failure. The inevitable focus on sudden death victims and transplant recipients may, however, have created a skewed perspective of a genetic disease. We hypothesized that unbiased representation of the spectrum of disease expression in arrhythmogenic right ventricular dysplasia/cardiomyopathy would require in vivo assessment of families in a genetically heterogeneous population. Methods and Results— A cohort of 200 probands and relatives satisfying task force or modified diagnostic criteria for arrhythmogenic right ventricular dysplasia/cardiomyopathy underwent comprehensive clinical evaluation. Desmosomal mutations were identified in 39 individuals from 20 different families. Indices of structural severity correlated with advancing age and were increased in long-term endurance athletes. Fulfillment of modified criteria indicated phenotypically mild disease, whereas asymptomatic status did not. In >80%, ECG, rhythm monitoring, and/or gadolinium-enhanced cardiovascular magnetic resonance were suggestive of LV involvement, the extent of which often was marked among individuals with chain-termination mutations and/or desmoplakin disease. Three patterns of disease expression were identified: (1) classic , with isolated right ventricular disease or LV involvement in association with significant right ventricular impairment; (2) left dominant , with early and prominent LV manifestations and relatively mild right-sided disease; and (3) biventricular , characterized by parallel involvement of both ventricles. Conclusions— LV involvement in arrhythmogenic right ventricular dysplasia/cardiomyopathy may precede the onset of significant right ventricular dysfunction. Recognition of disease variants with early and/or predominant LV involvement supports adoption of the broader term arrhythmogenic cardiomyopathy.