Arrhythmogenic Right Ventricular Cardiomyopathy Heart Research Articles

A 3D high resolution MRI method for the visualization of cardiac fibro-fatty infiltrations

Modifications of the myocardial architecture can cause abnormal electrical activity of the heart. Fibro-fatty infiltrations have been implicated in various cardiac pathologies associated with arrhythmias and sudden cardiac death, such as arrhythmogenic right ventricular cardiomyopathy (ARVC). Here, we report the development of an MRI protocol to observe these modifications at 9.4 T. Two fixed ex vivo human hearts, one healthy and one ARVC, were imaged with an Iterative decomposition with echo asymmetry and least-square estimations (IDEAL) and a magnetization transfer (MT) 3D sequences. The resulting fat fraction and MT ratio (MTR) were analyzed and compared to histological analysis of the three regions (“ARVC triangle”) primarily involved in ARVC structural remodeling. In the ARVC heart, high fat content was observed in the “ARVC triangle” and the superimposition of the MTR and fat fraction allowed the identification of fibrotic regions in areas without the presence of fat. The healthy heart exhibited twice less fat than the ARVC heart (31.9%, 28.7% and 1.3% of fat in the same regions, respectively). Localization of fat and fibrosis were confirmed by means of histology. This non-destructive approach allows the investigation of structural remodeling in human pathologies where fibrosis and/or fatty tissue infiltrations are expected to occur.

Integrin β1D Deficiency-Mediated RyR2 Dysfunction Contributes to Catecholamine-Sensitive Ventricular Tachycardia in Arrhythmogenic Right Ventricular Cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. Using protein mass spectrometry analyses, we identified that integrin β1 is downregulated in ARVC hearts without changes to Ca2+-handling proteins. As adult cardiomyocytes express only the β1D isoform, we generated a cardiac specific β1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin β1D loss on function in the heart in vivo and in vitro. Integrin β1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin β1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, β1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca2+ handling in β1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin β1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal-regulated kinase 1 and 2)-fibronectin-ubiquitin/lysosome pathway. Our data suggest that integrin β1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

Comprehensive Myocardial Proteogenomics Profiling Reveals C/EBPα as the Key Factor in the Lipid Storage of ARVC.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is hereditary cardiomyopathy characterized by the fibro-fatty replacement of the myocardium. A small number of noncomprehensive profiling studies based on human cardiac tissues have been conducted and reported; consequently, ARVC's gene expression pattern characteristics remain largely undocumented. Our study applies large-scaled, quantitative proteomics based on TMT-labeled LC-MS/MS to analyze the left and right ventricular myocardium of four ARVC and four DCM explanted hearts to compare them with normal hearts. Our objective is to reveal the characteristic proteome pattern in ARVC compared with DCM as well as nondiseased heart. We also conducted the RNA sequencing of 10 right ventricles from ARVC hearts paired with four nondiseased donor hearts to validate the proteome results. In a manner similar to that of the well-defined DCM heart failure model, the ARVC model demonstrates the downregulation of mitochondrial function proteins and the effects of many heart failure regulators such as TGFB, RICTOR, and KDM5A. In addition, the inflammatory signaling, especially the complement system, was activated much more severely in ARVC than in DCM. Our most significant discovery was the lipid metabolism reprogramming of both ARVC ventricles in accordance with the upregulation of lipogenesis factors such as FABP4 and FASN. We identified the key upstream regulator of lipogenesis as C/EBPα. Transcriptome profiling verified the consistency with proteome alterations. This comprehensive proteogenomics profiling study reveals that an activation of C/EBPα, along with the upregulation of its lipogenesis targets, accounts for lipid storage and acts as a hallmark of ARVC.

The TMEM43 Newfoundland mutation p.S358L causing ARVC-5 was imported from Europe and increases the stiffness of the cell nucleus

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetic condition caused predominantly by mutations within desmosomal genes. The mutation leading to ARVC-5 was recently identified on the island of Newfoundland and caused by the fully penetrant missense mutation p.S358L in TMEM43. Although TMEM43-p.S358L mutation carriers were also found in the USA, Germany, and Denmark, the genetic relationship between North American and European patients and the disease mechanism of this mutation remained to be clarified. We screened 22 unrelated ARVC patients without mutations in desmosomal genes and identified the TMEM43-p.S358L mutation in a German ARVC family. We excluded TMEM43-p.S358L in 22 unrelated patients with dilated cardiomyopathy. The German family shares a common haplotype with those from Newfoundland, USA, and Denmark, suggesting that the mutation originated from a common founder. Examination of 40 control chromosomes revealed an estimated age of 1300-1500 years for the mutation, which proves the European origin of the Newfoundland mutation. Skin fibroblasts from a female and two male mutation carriers were analysed in cell culture using atomic force microscopy and revealed that the cell nuclei exhibit an increased stiffness compared with TMEM43 wild-type controls. The German family is not affected by a de novo TMEM43 mutation. It is therefore expected that an unknown number of European families may be affected by the TMEM43-p.S358L founder mutation. Due to its deleterious clinical phenotype, this mutation should be checked in any case of ARVC-related genotyping. It appears that the increased stiffness of the cell nucleus might be related to the massive loss of cardiomyocytes, which is typically found in ventricles of ARVC hearts.

Reduced sodium channels in human ARVC

In recent years, the relatively uncommon disorder of arrhythmogenic right ventricular cardiomyopathy (ARVC) has received a lot of attention from basic and clinical investigators. Histological characterization of ARVC hearts is notable for patchy fibrofatty replacement of the myocardium, and the syndrome can result in both heart failure and ventricular arrhythmias. Slightly more than half of the patients with ARVC have mutations in desmosomal proteins1 residing in the intercalated disc that connects adjacent cardiomyocytes.

Myocardial transcriptome analysis of human arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.

Molecular changes in the heart of a severe case of arrhythmogenic right ventricular cardiomyopathy caused by a desmoglein-2 null allele

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder caused by mutations in desmosomal genes. It is often associated with life-threatening arrhythmias. Some affected individuals develop progressive heart failure and may require cardiac transplantation. The explanted heart of a young adult with end-stage heart failure due to a null allele in desmoglein-2 was studied at macroscopic, microscopic, and molecular level. Myocardial samples were probed for junctional localization of desmosomal components and the gap junction protein connexin43 by immunohistochemical staining. In addition, the protein content of desmosomal and adherens junction markers as well as connexin43 was assessed by Western blotting. Histological analysis confirmed ARVC. Despite the loss of specific immunoreactive signal for desmosomal components at the cardiac intercalated disks (shown for plakoglobin, desmoplakin, and plakophilin-2), these proteins could be detected by Western blotting. Only for desmoglein-2, desmocollin-2, and plakoglobin were reduced protein levels observed. Adherens junction proteins were not affected. Lower phosphorylation levels were observed for connexin43; however, localization of the gap junction protein displayed regional differences. At the molecular level, disease progression was more severe in the right ventricle compared to the left ventricle. Our data suggest that, in the ARVC heart, plakoglobin is mainly redistributed from the junctions to other cellular pools and that protein degradation only plays a secondary role. Homogenous changes in the phosphorylation status of connexin43 were observed in multiple ARVC samples, suggesting that this might be a general feature of the disease.

Proteomic analysis reveals significant elevation of heat shock protein 70 in patients with chronic heart failure due to arrhythmogenic right ventricular cardiomyopathy

As proteins are the ultimate biological determinants of phenotype of disease, we screened altered proteins associated with heart failure due to arrhythmogenic right ventricular cardiomyopathy (ARVC) to identify biomarkers potential for rapid diagnosis of heart failure. By 2-dimensional gel electrophoresis and mass spectrometry, we identified five commonly altered proteins with more than 1.5 fold changes in eight ARVC failing hearts using eight non-failing hearts as reference. Noticeably, one of the altered proteins, heat shock protein 70 (HSP70), was increased by 1.64 fold in ARVC failing hearts compared with non-failing hearts. The increase of cardiac HSP70 was further validated by Western blot, immunochemistry, and enzyme-linked immunosorbent assay (ELISA) in failing hearts due to not only ARVC, but also dilated (DCM, n = 18) and ischemic cardiomyopathy (ICM, n = 8). Serum HSP70 was also observed to be significantly increased in heart failure patients derived from the three forms of cardiomyopathies. In addition, we observed hypoxia/serum depletion stimulation induced significantly elevation of intracellular and extracellular HSP70 in cultured neonatal rat cardiomyocytes. For the first time to our knowledge, we revealed and clearly demonstrated significant up-regulation of cardiac and serum HSP70 in ARVC heart failure patients. Our results indicate that elevated HSP70 is the common feature of heart failure due to ARVC, DCM, and ICM, which suggests that HSP70 may be used as a biomarker for the presence of heart failure due to cardiomyopathies of different etiologies and may hold diagnostic/prognostic potential in clinical practice.

A potential link between peroxisome proliferator-activated receptor signalling and the pathogenesis of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by major fibro-fatty replacement of the right ventricle (RV). We hypothesized that changes in peroxisome proliferator-activated receptor (PPAR) signalling contributed to myocardium fatty accumulation and contractile dysfunction in ARVC. Real-time quantitative reverse transcriptase-polymerase chain reaction and western blotting were used to assess cardiac expression of PPARalpha and gamma and two of their downstream target genes--medium-chain acyl-CoA dehydrogenase (MCAD) and phosphoenolpyruvate carboxykinase (PEPCK)--in both RV and left ventricle (LV) from five controls and five ARVC patients. In vitro motility assays were used to analyse functional properties of myosin. In the RV, sliding velocity was nearly two-fold lower in ARVC than in controls, whereas a 10% reduction in velocity values was noted between ARVC and non-failing myocardium in the LV. In controls, PPARalpha and MCAD mRNA and protein levels were higher in the RV compared with the LV. In ARVC, the expression of PPARalpha and MCAD mRNA and/or proteins was decreased in both RV and LV. RV from ARVC was also characterized by a dramatic activation of the PPARgamma pathway, as attested by the increase in PPARgamma mRNA and protein (500 and 270%, respectively, each P < 0.001) and by the induction of PEPCK gene. In contrast, the LV of ARVC heart exhibited no changes in the expression of the PPARgamma regulatory pathway compared with control. ARVC is associated with major disturbances in the PPARalpha and PPARgamma signalling pathway in the RV that may contribute to intracellular lipid overload and severe myosin dysfunction.

Abstract 5934: Arrhythmogenic Right Ventricular Cardiomyopathy Due to Desmosomal Gene Mutations Is a Progressive Biventricular Disease

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease characterized by a gradual loss of myocytes with fibro-fatty replacement. Mutations of gene encoding desmosomal proteins have been demonstrated in up to 50% of probands. The aim of this study was to assess the morphologic spectrum and the relative role of viruses on genotyped ARVC hearts. Design: Fourteen ARVC hearts (10 M/4 F, age range 7–64, mean 30 yrs), coming from sudden death (8), cardiac transplantation (4), heart failure (1) and extracardiac death (1) were investigated. Familial recurrence of ARVC was ascertained in 12 (86%). Genetic screening identified pathogenetic mutations in plakophilin-2 (5), desmoplakin (4), desmoglein-2 (2) and plakoglobin (3). Five hearts from patients with in ryanodine receptor 2 (Ryr2) gene mutations were also investigated. Morphopathologic investigation consisted of gross examination, histology, immunohistochemistry and molecular pathology (PCR) for cardiotropic viruses. Result: Cardiac involvement was biventricular in all but 1 case, a 7 year old girl who died of extracardiac causes and presented a structurally normal heart. Ventricular myocardium abnormalities consisted of fibro-fatty replacement in 12 cases and of subepicardial myocyte necrosis with acute inflammation and granulation tissue in a 15 year old boy who died suddenly. Inflammatory infiltrates were evident in 93% of cases, either diffuse (23%) or focal (77%), and mostly consisted of T-lymphocytes and macrophages. Molecular investigation was negative in all but one with Hepatitis C virus (7%). Ryr2 gene hearts showed only mild fatty infiltration confined to the antero-apical wall of the right ventricle. Conclusion: ARVC hearts from patients with desmosomal gene mutations are characterized by biventricular myocardial atrophy and fibro-fatty replacement. In early adolescence, the phenotype can be either absent or consistent with acute myocyte damage without fibro-fatty replacement, thus confirming the progressive nature of the disease. While myocarditis is a usual feature, viral genome is exceptionally detected in the myocardium as to support the reactive nature of inflammation. The pathologic features of Ryr2 hearts are not in keeping with ARVC.