Angeliki Asimaki

Clinical and Genetic Characterization of Families With Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Provides Novel Insights Into Patterns of Disease Expression

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.

A New Diagnostic Test for Arrhythmogenic Right Ventricular Cardiomyopathy

BACKGROUND The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low. METHODS To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples. RESULTS We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum. CONCLUSIONS Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Associated with Mutations in the Desmosomal Gene Desmocollin-2

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited myocardial disorder associated with arrhythmias, heart failure, and sudden death. To date, mutations in four genes encoding major desmosomal proteins (plakoglobin, desmoplakin, plakophilin-2, and desmoglein-2) have been implicated in the pathogenesis of ARVD/C. We screened 77 probands with ARVD/C for mutations in desmocollin-2 (DSC2), a gene coding for a desmosomal cadherin. Two heterozygous mutations--a deletion and an insertion--were identified in four probands. Both mutations result in frameshifts and premature truncation of the desmocollin-2 protein. For the first time, we have identified mutations in desmocollin-2 in patients with ARVD/C, a finding that is consistent with the hypothesis that ARVD/C is a disease of the desmosome.

Clinical Expression of Plakophilin-2 Mutations in Familial Arrhythmogenic Right Ventricular Cardiomyopathy

Background— Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disorder characterized by loss of cardiomyocytes and their replacement by adipose and fibrous tissue. It is considered a disease of cell adhesion because mutations in desmosomal genes, desmoplakin and plakoglobin, have been implicated in the pathogenesis of ARVC. In a recent report, mutations in plakophilin-2, a gene highly expressed in cardiac desmosomes, have been shown to cause ARVC. Methods and Results— We investigated 100 white patients with ARVC for mutations in plakophilin-2. Nine different mutations were identified by direct sequencing in 11 cases. Five of these mutations are novel (A733fsX740, L586fsX658, V570fsX576, R413X, and P533fsX561) and predicted to cause a premature truncation of the plakophilin-2 protein. Family studies showed incomplete disease expression in mutation carriers and identified a number of individuals who would be misdiagnosed with the existing International Task Force and modified diagnostic criteria for ARVC. Conclusions— In this study, we provide new evidence that mutations in the desmosomal plakophilin-2 gene can cause ARVC. A systematic clinical evaluation of mutation carriers within families demonstrated variable phenotypic expression, even among individuals with the same mutation, and highlighted the need for a more accurate set of diagnostic criteria for ARVC.

A Novel Dominant Mutation in Plakoglobin Causes Arrhythmogenic Right Ventricular Cardiomyopathy

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.

Phospholamban R14del mutation in patients diagnosed with dilated cardiomyopathy or arrhythmogenic right ventricular cardiomyopathy: evidence supporting the concept of arrhythmogenic cardiomyopathy

To investigate whether phospholamban gene (PLN) mutations underlie patients diagnosed with either arrhythmogenic right ventricular cardiomyopathy (ARVC) or idiopathic dilated cardiomyopathy (DCM).

Identification of a New Modulator of the Intercalated Disc in a Zebrafish Model of Arrhythmogenic Cardiomyopathy

Drug screening in a zebrafish model of arrhythmogenic cardiomyopathy identified a small molecule that remodels the intercalated disc. Fishing for Heart Healing When Judy Garland sings “Thump, thump, thump went my heart strings,” she’s celebrating a new love connection. But it remains unclear why patients with arrhythmogenic cardiomyopathy (ACM) suffer frequent heart arrhythmias. Asimaki et al. used a zebrafish model of ACM to illuminate disease mechanisms and discovered a potential drug, SB216763, that suppressed the disease phenotype in the fish model. The authors also observed SB216763-reversible, abnormal cardiac remodeling in cardiac myocytes derived from rats that carried an ACM-related mutation and from induced pluripotent stem cells from two ACM probands. These observations pinpoint aberrant trafficking of cardiac proteins as a central mechanism underlying ACM cardiomyocyte injury and electrical abnormalities. Arrhythmogenic cardiomyopathy (ACM) is characterized by frequent cardiac arrhythmias. To elucidate the underlying mechanisms and discover potential chemical modifiers, we created a zebrafish model of ACM with cardiac myocyte–specific expression of the human 2057del2 mutation in the gene encoding plakoglobin. A high-throughput screen identified SB216763 as a suppressor of the disease phenotype. Early SB216763 therapy prevented heart failure and reduced mortality in the fish model. Zebrafish ventricular myocytes that expressed 2057del2 plakoglobin exhibited 70 to 80% reductions in INa and IK1 current densities, which were normalized by SB216763. Neonatal rat ventricular myocytes that expressed 2057del2 plakoglobin recapitulated pathobiological features seen in patients with ACM, all of which were reversed or prevented by SB216763. The reverse remodeling observed with SB216763 involved marked subcellular redistribution of plakoglobin, connexin 43, and Nav1.5, but without changes in their total cellular content, implicating a defect in protein trafficking to intercalated discs. In further support of this mechanism, we observed SB216763-reversible, abnormal subcellular distribution of SAP97 (a protein known to mediate forward trafficking of Nav1.5 and Kir2.1) in rat cardiac myocytes expressing 2057del2 plakoglobin and in cardiac myocytes derived from induced pluripotent stem cells from two ACM probands with plakophilin-2 mutations. These observations pinpoint aberrant trafficking of intercalated disc proteins as a central mechanism in ACM myocyte injury and electrical abnormalities.

Severe cardiac phenotype with right ventricular predominance in a large cohort of patients with a single missense mutation in the DES gene

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.

Electrophysiological abnormalities precede overt structural changes in arrhythmogenic right ventricular cardiomyopathy due to mutations in desmoplakin-A combined murine and human study

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.

Altered Desmosomal Proteins in Granulomatous Myocarditis and Potential Pathogenic Links to Arrhythmogenic Right Ventricular Cardiomyopathy

Background— Immunoreactive signal for the desmosomal protein plakoglobin (&ggr;-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-&agr; (TNF-&agr;), 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-&agr; and elevated levels of serum inflammatory mediators, including IL-6R, IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1&bgr;, in patients with ARVC (all P<0.0001 compared with controls). 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.