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Journal of Investigative Medicine:
doi: 10.231/JIM.0b013e3181c5e631
EB Symposium Manuscripts

Arrhythmogenic Right Ventricular Cardiomyopathy: New Insights Into Disease Mechanisms and Diagnosis

Saffitz, Jeffrey E. MD, PhD*; Asimaki, Angeliki PhD*; Huang, Hayden PhD†

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From the *Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA; and †Department of Biomedical Engineering, Columbia University, New York, NY.

Received September 9, 2009, and in revised form October 6, 2009.

Accepted for publication October 6, 2009.

Reprints: Jeffrey E. Saffitz, MD, PhD, Department of Pathology, Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA 02115. E-mail:

Supported by the Kenneth M. Rosen Fellowship in Cardiac Pacing and Electrophysiology from the Heart Rhythm Society (A. A.) and by the Lerner Fund (H. H.), and the symposium was supported in part by a grant from the National Center for Research Resources (R13 RR023236).

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Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder characterized by the early occurrence of serious tachyarrhythmias often out of proportion to the extent of structural changes and contractile derangement. Approximately 40% of patients with ARVC 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 nuclear signaling molecules. It has been proposed that mutations in desmosomal proteins implicated in ARVC may perturb the normal balance of protein in junctions and the cytosol which, in turn, could promote dysregulated gene expression circumventing the normal controls of Wnt signaling pathways. This review highlights recent advances in understanding the pathogenesis of ARVC and presents evidence, suggesting that the disease is caused by a combination of altered cellular biomechanical behavior and altered signaling.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a primary heart muscle disorder. The cardinal feature that distinguishes it from other heart muscle diseases associated with arrhythmias and sudden death (such as dilated or hypertrophic cardiomyopathies) is the early occurrence of arrhythmias out of proportion to the extent of structural changes and contractile derangement.1 In fact, ARVC is the most arrhythmogenic human heart disease known. Approximately 40% of ARVC patients who receive implantable cardiac defibrillators experience an appropriate shock within 2 years, a far greater rate than in patients with hypertrophic cardiomyopathy or ion channelopathies such as the long QT syndrome (from the North American ARVD Registry). Classically, we think of ARVC as a disease in which injured right ventricular myocardium is replaced by fibrofatty scar tissue, but although this pathological change may become especially prominent as the disease progresses, it is not unusual for patients to manifest life-threatening arrhythmias with only minimal accumulation of fat and fibrous tissue.

Approximately 40% of patients with ARVC have one or more mutations in genes encoding desmosomal proteins.2 Desmosomes are intercellular adhesion junctions, which, in cardiac myocytes, reside within intercalated disks. They contain adhesion molecules of the cadherin family (desmogleins and desmocollins), which span the cell membrane and bind in the extracellular space to connect adjacent cells, and linker proteins of the plakin and catenin families (desmoplakin, plakophilins, and plakoglobin), which form intracellular assemblies linking the desmosomal cadherins to intermediate filaments of the cytoskeleton.3 Desmosomes are especially abundant in tissues that bear a high mechanical load such as heart and skin. It is not surprising, therefore, that human diseases related to mutations in desmosomal proteins are manifest clinically as cardiac or cutaneous diseases, with phenotypic expression determined by the specific tissue distribution of the mutant protein and the severity of the mutation. Mutations implicated in the pathogenesis of arrhythmogenic cardiomyopathy have been identified in genes encoding all recognized desmosomal proteins.2 Some of these proteins (particularly plakoglobin) fulfill roles both as structural proteins in cell-cell adhesion junctions and as nuclear signaling molecules. Under basal conditions, plakoglobin is found mainly in desmosomes. The cytosolic concentration is kept low because plakoglobin is efficiently targeted for proteasomal degradation by a regulatory mechanism involving the canonical Wnt signaling pathway.3 When this pathway is activated, targeted degradation of plakoglobin is inhibited, causing intracellular accumulation, increased nuclear transport, and subsequent changes in gene expression.3 It has been proposed that mutations in desmosomal proteins implicated in ARVC may perturb the normal balance of protein in junctions and the cytosol, which, in turn, could promote dysregulated gene expression circumventing the normal controls of Wnt signaling pathways. Although this question has not been studied in detail in diseased human myocardium, studies in animal models and expression systems in vitro have documented increased nuclear and cytoplasmic pools of mutant desmosomal proteins.3,4 Thus, current evidence suggests that the pathogenesis of arrhythmogenic cardiomyopathy is related to a combination of altered cellular biomechanical behavior and altered signaling.

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To gain initial insights into mechanisms by which mutations in desmosomal proteins cause ARVC in patients, we have analyzed diseased human myocardium in an effort to answer 3 main questions. First, ARVC is typically caused by dominant mutations.2 Thus, we have asked whether the mutant proteins are expressed and, if so, whether they reside in junctions or accumulate in the cytoplasm. Second, desmosomes consist of molecular assemblies dependent on complex binding interactions among multiple proteins. Thus, we have asked whether a mutation in a single desmosomal protein can affect the distribution of other (nonmutant) desmosomal proteins. And third, it is well known that normal cell-cell coupling at gap junctions depends on normal intercellular adhesion.5 Thus, we have investigated whether mutations in proteins of cell-cell adhesion junctions can affect the structure and function of electrical connections at gap junctions and, thereby, potentially contribute to the highly arrhythmogenic phenotype characteristic of this spectrum of cardiomyopathies. Most of the worldwide resource of tissue from patients with ARVC consists of formalin-fixed, paraffin-embedded autopsy or endomyocardial biopsy material. Although one cannot do extensive "experiments" on this material, there is still much to be learned from a rigorous analysis to define the molecular pathology of the disease. Our principal approach has been to use immunohistochemistry to characterize the distribution of desmosomal and gap junction proteins at cardiac myocyte intercalated disks in tissues from patients with ARVC. We designed this approach specifically to show bright immunoreactive signal at intercalated disks where antigen concentrations are normally very high. It is not designed to show protein in other compartments such as the cytosol or nucleus, in which antigen concentrations are much lower. Thus, we developed this method to determine whether the amount of signal at cell-cell junctions is normal or not. Diminished signal intensity indicates that less than the normal amount of protein is present at junctions. However, absent signal does not mean that no protein is present, but only that the amount is diminished to below the detectable level.

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Altered Subcellular Distribution of Plakoglobin in ARVC

Our initial studies were focused on Naxos disease and Carvajal syndrome, rare forms of arrhythmogenic cardiomyopathy associated with changes in hair and skin.6,7 These cardiocutaneous syndromes are caused by recessive mutations of plakoglobin and desmoplakin, respectively. In both diseases, frameshifts lead to premature termination and truncation of C-terminal portions of the proteins.8,9 In studies of ventricular myocardium from patients with Naxos disease, we observed that the mutant protein, plakoglobin, failed to localize normally at intercellular junctions, whereas Western blot analysis showed that it was clearly expressed in the tissue.6 It was possible to definitively identify mutant plakoglobin because the truncated protein migrated more rapidly on polyacrylamide gels.6 These results indicate that although the mutant protein is expressed, it fails to localize at junctions and may, therefore, accumulate in the cytosolic pool. A similar type of immunohistochemical analysis in Carvajal syndrome showed not only that the mutant protein, desmoplakin, fails to localize at intercalated disks but also that its binding partner, plakoglobin, fails to localize.7 The key insight gained here was that a mutation in a single desmosomal protein may perturb the subcellular distribution of another protein that is not genetically altered.

We have now performed immunohistochemical analysis of myocardium from more than 30 patients with clinically and/or pathologically documented ARVC (without associated cutaneous abnormalities) many of whom have been shown to have dominant mutations in a variety of desmosomal genes.10 Our observations have led to important insights that bear directly on mechanisms of disease pathogenesis in patients. First, we have found that a single gene mutation leads to various patterns of altered localization of desmosomal proteins even when the mutation affects only a single allele. This observation is consistent with the idea that the mutant protein alters binding interactions within the desmosome such that one or more desmosomal proteins may redistribute from junctional to cytosol compartments and, potentially, change nuclear signaling and gene expression patterns. Second, we have found that in nearly every case of ARVC, signal for the intracellular linker protein, plakoglobin, is diminished at intercalated disks.10 This remarkably consistent finding occurred in the disease caused by mutations in desmoplakin (3 different mutations), plakophilin-2 (2 different mutations), plakoglobin (2 different mutations), and desmoglein-2 (2 different mutations) (Fig. 1). It also occurred in several cases of pathologically documented ARVC in which there were no known mutations in desmosomal genes. Reduced plakoglobin signal is apparently highly specific for ARVC. It did not occur in 15 cases of end-stage heart disease caused by ischemic, dilated, or hypertrophic cardiomyopathy (n = 5 for each) (Fig. 2).10 These observations are of potential importance for 2 reasons. First, it forms the basis for a new diagnostic test for ARVC, which we are currently validating and implementing. Second, it implicates reduced localization of plakoglobin at intercalated disks as a fundamental feature of ARVC in patients and suggests that redistribution of plakoglobin from junctions to intracellular pools is part of a final common pathway in disease pathogenesis.

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Of course, it must be acknowledged that we still do not know the cause of ARVC in more than 50% of the patients without desmosomal mutations. Whether redistribution of plakoglobin from junctions to intracellular pools occurs in these patients and plays a role in disease pathogenesis remains unknown and requires additional study.

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Gap Junction Remodeling in ARVC

An extensive literature exists on the dependence of cell-cell coupling at gap junctions on normal cell-cell adhesion (see Saffitz5 for a review). This dependence seems to be especially critical in the heart in which unusually large gap junctions, required for safe conduction, must be maintained under physiological conditions of cyclical contractile activity. We were the first to report that abnormalities in intercellular adhesion caused by mutations in desmosomal proteins may promote remodeling of gap junctions, which, in turn, could alter conduction and potentially account for the arrhythmogenic phenotype in this disease (this idea was originally suggested to us by Guy Fontaine).6,7 We first observed marked remodeling of gap junctions in Naxos disease and Carvajal syndrome,6,7 but now, in more recent studies of dominant forms of ARVC associated with mutations in various desmosomal proteins, we observed marked reduction in Cx43 immunoreactive signal at intercalated disks in every case (Fig. 1).10 We also found that gap junction remodeling occurs diffusely in ARVC, including regions of the left ventricle and interventricular septum, which do not exhibit the classic pathological features of myocyte degeneration and fibrofatty replacement.10 These results indicate that remodeling of gap junctions is a consistent feature of ARVC. Of course, diminished Cx43 signal at intercalated disks is not specific for ARVC-it has been well documented in ischemic and nonischemic forms of heart muscle disease. However, reduced Cx43 expression in these cardiomyopathies is most apparent in advanced disease in which there has been considerable structural remodeling. In contrast, reduced gap junction density can occur early in ARVC, before significant structural changes become manifest in the right ventricle.6,10 This is a potentially important distinction that hints at disease-specific mechanisms. It also suggests that gap junction remodeling is mechanistically linked to the fundamental disease pathway in ARVC.

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How Do Mutations in Desmosomal Proteins Affect Cell Biomechanical Behavior?

We previously reported a novel dominant mutation in plakoglobin (118_119insGCA), which is predicted to lead to insertion of an extra serine residue within the N-terminal domain of the protein (S39_K40insS).4 We found that the mutant plakoglobin, but not the wild type protein, failed to localize normally at cell-cell junctions and accumulated in both the cytoplasm and nucleus in human embryonic kidney (HEK) 293 cell lines transfected to stably express either wild type or S39_K40insS plakoglobin.4 We also analyzed HEK cells that expressed the other known disease-causing mutation in plakoglobin (2057del2), which is responsible for the recessive cardiocutaneous syndrome Naxos disease. We had previously observed in the human disease that 2057del2 also prevented normal accumulation of plakoglobin at cell-cell junctions despite the fact that the protein was abundantly expressed.6 Accordingly, we used HEK cells expressing 2 distinct ARVC-causing mutations in plakoglobin (S39_K40insS and 2057del2) to ask whether mutant plakoglobin alters cellular biomechanical behavior. We observed that expression of either mutant form of plakoglobin accelerated wound healing in the classic in vitro assay related not to a change in cell proliferation rate but to increased cell motility.11 To determine whether expression of mutant plakoglobin altered cellular responses to mechanical perturbation, we grew cells on deformable silicone membranes and subjected them to linear, pulsatile stretch (to 110% of resting length at 3 Hz) for 1 or 4 hours. As previously observed in cardiac myocytes,12 the amount of signal for plakoglobin and Cx43 at cell junctions increased significantly after stretch in cells expressing wild type plakoglobin, whereas these responses were significantly blunted in cells expressing either mutant form of plakoglobin.11 Interestingly, the amount of Cx43 at cell-cell junctions was significantly reduced under basal (nonstretched) conditions in cells expressing mutant plakoglobin, consistent with our previous observations in ARVC patients.6,7,10 In additional studies, we used magnetic micromanipulation assays and found that S39_K40inS, but not 2057del2, significantly diminished cell stiffness.11 In contrast, a robust deformation-drag assay revealed a dramatic reduction in the strength of cell-cell adhesion in cells expressing 2057del2 but no change with S39_K40inS.11 These results were confirmed in independent dispase dissociation assays. Taken together, these novel results suggest that myocardial injury in Naxos disease may arise from loss of cell-cell adhesion, whereas increased sensitivity to intracellular mechanosignaling cascades may occur with S39_K40insS. However, whether altered biomechanical behavior leads to increased arrhythmogenesis is unknown. Additional studies in cardiac myocytes and informative animal models will be required to elucidate potential mechanistic relationships.

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Arrhythmogenic right ventricular cardiomyopathy has an unusually dramatic arrhythmogenic phenotype, which is manifest early in the natural history of the disease, often preceding the development of significant ventricular remodeling or contractile dysfunction. Although there has been important progress in identifying mutations in desmosomal genes that lead to ARVC, much less is known about how the mutant proteins cause the disease. One leading hypothesis is that abnormal cell-cell adhesion injures cardiac myocytes and promotes cell death and subsequent replacement by fibrofatty tissue. Such a mechanism almost certainly plays a role. However, desmosomal proteins may fulfill dual roles as structural proteins in adhesion junctions and as signaling molecules, which can inhibit Wnt signaling and thereby modulate pathological gene expression, promote cardiac myocyte apoptosis, and perhaps mediate expression of a fibrogenic and/or adipogenic phenotype. Either or both mechanisms could lead to gap junction remodeling as an early manifestation in ARVC, but little is actually known about the responsible mechanism(s). We have discovered that the desmosomal protein plakoglobin (aka γ-catenin) fails to localize in cell-cell junctions in a most cases of ARVC regardless of the specific mutation involved or even when no mutation can be identified. This suggests a final common pathway in which desmosomal instability, caused by mutations in one or more desmosomal proteins, causes subcellular redistribution of plakoglobin, which plays a pivotal role in altered signaling pathways. This unifying hypothesis provides a novel, testable explanation for the clinical observation that ARVC patients often experience acute exacerbations after intense exercise.

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1. Muthappan P, Calkins H. Arrhythmogenic right ventricular dysplasia. Prog Cardiovasc Dis. 2008;51:31-43.

2. Sen-Chowdhry S, Syrris P, McKenna WJ. Genetics of right ventricular cardiomyopathy. J Cardiovasc Electrophysiol. 2005;16:927-935.

3. Zhurinsky J, Shtutman M, Ben-Ze'ev A. Plakoglobin and β-catenin: protein interactions, regulation and biological roles. J Cell Sci. 2000;113:3127-3139.

4. Asimaki A, Syrris P, Wichter T, et al. A novel dominant mutation in plakoglobin causes arrhythmogenic right ventricular cardiomyopathy. Am J Human Genet. 2007;81:964-973.

5. Saffitz JE. Dependence of electrical coupling on mechanical coupling in cardiac myocytes. In: Thiene G, Dessina AC, eds. Advances in Cardiovascular Medicine. Universitá degli Studi di Padova, Padua, Italy, 2003:15-28.

6. Kaplan SR, Gard JJ, Protonotarios N, et al. Remodeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm. 2004;1:3-11.

7. Kaplan SR, Gard JJ, Carvajal-Huerta L, et al. Structural and molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol. 2004;13:26-32.

8. McKoy G, Protonotarios N, Crosby A, et al. Identification of a deletion in plakoglobin in arrhythmogenic right ventricular cardiomyopathy with palmoplantar keratoderma and woolly hair (Naxos disease). Lancet. 2000;355:2119-2124.

9. Norgett EE, Hatsell SJ, Carvajal-Huerta K, et al. Recessive mutation in desmoplakin disrupts desmoplakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma. Hum Mol Gen. 2000;9:2761-2766.

10. Asimaki A, Tandri H, Huang H, et al. A new diagnostic test for arrhythmogenic right ventricular cardiomyopathy. N Eng J Med. 2009;360:1075-1084.

11. Huang H, Asimaki A, Lo D, et al. Disparate effects of different mutations in plakoglobin on cell mechanical behavior. Cell Motil Cytoskel. 2008;65:964-978.

12. Yamada K, Green KG, Samarel AM, et al. Distinct pathways regulate expression of cardiac electrical and mechanical junction proteins in response to stretch. Circ Res. 2005;97:346-353.


desmosome; plakoglobin; gap junctions; arrhythmias; biomechanical behavior

© 2009 American Federation for Medical Research


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