Mutations In Desmosomal Proteins Research Articles

Arrhythmogenic cardiomyopathy and abnormalities of cell-to-cell coupling

Arrhythmogenic cardiomyopathy is characterized by a high incidence of ventricular tachyarrhythmias, which often occur early in the disease before significant ventricular remodeling or contractile dysfunction develops. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is the most common clinicopathological form of this group of diseases, but left ventricular and biventricular forms have been increasingly recognized. Approximately a third of patients with arrhythmogenic cardiomyopathy have mutations in genes encoding proteins of the desmosome, cell-to-cell adhesion organelles located at cardiac myocyte intercalated disks. Using immunohistochemistry in heart biopsies or autopsy specimens, we have observed diminished localization at intercalated disks of specific patterns of desmosomal proteins in a great majority of patients with ARVC. Because some of these proteins play roles as both structural proteins in cell-to-cell mechanical junctions and as signaling molecules, the pathogenesis of arrhythmogenic cardiomyopathy is probably related to both derangements in cell-to-cell adhesion and altered nuclear signaling. We have also observed diminished immunoreactive signal for the gap junction protein Cx43 at intercalated disks in virtually all patients with ARVC. Gap junction remodeling occurs diffusely in ARVC, and it appears early in the disease before significant degenerative changes of the myocardium have developed. Thus, mutations in desmosomal proteins may not only cause myocyte injury eventually leading to cell death and fibro-fatty tissue replacement but may also contribute to development of anatomic substrates of sudden death by remodeling gap junctions and altering electrical conduction.

Characterization of the molecular phenotype of two arrhythmogenic right ventricular cardiomyopathy (ARVC)-related plakophilin-2 (PKP2) mutations

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to mutations in desmosomal proteins, including plakophilin-2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations. The purpose of this study was to investigate the function and distribution of an ARVC-relevant PKP2 mutant where arginine at position 79 was replaced by a stop codon (R79x). Results were compared with those obtained with mutation 179fs (frameshift at position 179). Mutant constructs were introduced by adenoviral infection into neonatal rat ventricular myocytes in culture. Both mutant proteins failed to preferentially localize to sites of cell-cell apposition. Their expression did not disrupt localization of endogenous PKP2, connexin-43 (Cx43), or desmoplakin (DP). However, we observed reduced abundance of Cx43 after R79x expression. Early truncation of PKP2 at position 79 also prevented its physical interaction with both DP and Cx43. Finally, R79x expression correlated with loss of expression of HSP90, a protein relevant to cardiomyocyte apoptosis. These results provide the first observations of the cellular/molecular phenotype consequent to these PKP2 mutations and give insight into the possible cellular substrates that lead to ARVC.

Connexin43 Remodeling Caused by Inhibition of Plakophilin-2 Expression in Cardiac Cells

Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts. We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins. The results are discussed in the context of arrhythmogenic right ventricular cardiomyopathy, an inherited disease involving mutations in desmosomal proteins, including PKP2.

Plakophilin-2 missense mutations in arrhythmogenic right ventricular cardiomyopathy

Background Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited cardiac disorder characterized by life-threatening ventricular arrhythmias and fibrofatty replacement of myocardial tissue. Recent data suggest a dominant mode of inheritance in ARVD due to mutations in desmosomal proteins, plakophilin-2 (PKP2) in particular. We carried out a search for PKP2 mutations in the Finnish population representing a genetic isolate. Methods Mutations were detected by direct sequencing of PKP2 exons in 29 unrelated ARVD patients. Subcellular changes in ARVD associated with PKP2 mutations were searched for using immunohistochemistry and electron microscopy. Results We identified three PKP2 amino acid substitutions, absent in controls, in three (10%) cases. Two of them (Q62K and N613K) co-occurred in a patient with arrhythmia and structural changes of the heart. Visualized with plakophilin-2 antibodies, the intercalated disks in this compound heterozygous ARVD sample appeared wavier than in non-ARVD controls. Partial irregularities were occasionally seen in the organization and distribution of the cell-cell junctions. Relatives carrying one of these mutant alleles were phenotypically normal or showed only limited electrocardiographic (ECG) changes. The third substitution (Q59L) was detected in two ARVD probands with ventricular tachycardias, ECG abnormalities and right ventricular structural alterations. Conclusions We identified two novel plakophilin-2 missense mutations associated with 10% of ARVD, and a previously reported Q62K variant with a possible disease modifying role. The low prevalence of predominantly missense mutations may present population-specific differences in the pathogenesis of ARVD. Our preliminary data also suggest that ultrastructural cell junction abnormalities may associate with plakophilin-2 mutations.

Mutant Desmocollin-2 Causes Arrhythmogenic Right Ventricular Cardiomyopathy

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous heart-muscle disorder characterized by progressive fibrofatty replacement of right ventricular myocardium and an increased risk of sudden cardiac death. Mutations in desmosomal proteins that cause ARVC have been previously described; therefore, we investigated 88 unrelated patients with the disorder for mutations in human desmosomal cadherin desmocollin-2 (DSC2). We identified a heterozygous splice-acceptor-site mutation in intron 5 (c.631-2A-->G) of the DSC2 gene, which led to the use of a cryptic splice-acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed a requirement for dsc2 in the establishment of the normal myocardial structure and function, with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines, and associated myocardial contractility defects. These data identify DSC2 mutations as a cause of ARVC in humans and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis, and cardiac function.

Age- and Training-Dependent Development of Arrhythmogenic Right Ventricular Cardiomyopathy in Heterozygous Plakoglobin-Deficient Mice

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disorder that causes sudden death and right ventricular heart failure in the young. Clinical data suggest that competitive sports may provoke ARVC in susceptible persons. Genetically, loss-of-function mutations in desmosomal proteins (plakophilin, desmoplakin, or plakoglobin) have been associated with ARVC. To test the hypothesis that reduced desmosomal protein expression causes ARVC, we studied the cardiac effects of heterozygous plakoglobin deficiency in mice. Ten-month-old heterozygous plakoglobin-deficient mice (plakoglobin+/-) had increased right ventricular volume, reduced right ventricular function, and spontaneous ventricular ectopy (all P<0.05). Left ventricular size and function were not altered. Isolated, perfused plakoglobin+/- hearts had spontaneous ventricular tachycardia of right ventricular origin and prolonged right ventricular conduction times compared with wild-type hearts. Endurance training accelerated the development of right ventricular dysfunction and arrhythmias in plakoglobin+/- mice. Histology and electron microscopy did not identify right ventricular abnormalities in affected animals. Heterozygous plakoglobin deficiency provokes ARVC. Manifestation of the phenotype is accelerated by endurance training. This suggests a functional role for plakoglobin and training in the development of ARVC.

Suppression of canonical Wnt/ -catenin signaling by nuclear plakoglobin recapitulates phenotype of arrhythmogenic right ventricular cardiomyopathy

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is a genetic disease caused by mutations in desmosomal proteins. The phenotypic hallmark of ARVC is fibroadipocytic replacement of cardiac myocytes, which is a unique phenotype with a yet-to-be-defined molecular mechanism. We established atrial myocyte cell lines expressing siRNA against desmoplakin (DP), responsible for human ARVC. We show suppression of DP expression leads to nuclear localization of the desmosomal protein plakoglobin and a 2-fold reduction in canonical Wnt/beta-catenin signaling through Tcf/Lef1 transcription factors. The ensuing phenotype is increased expression of adipogenic and fibrogenic genes and accumulation of fat droplets. We further show that cardiac-restricted deletion of Dsp, encoding DP, impairs cardiac morphogenesis and leads to high embryonic lethality in the homozygous state. Heterozygous DP-deficient mice exhibited excess adipocytes and fibrosis in the myocardium, increased myocyte apoptosis, cardiac dysfunction, and ventricular arrhythmias, thus recapitulating the phenotype of human ARVC. We believe our results provide for a novel molecular mechanism for the pathogenesis of ARVC and establish cardiac-restricted DP-deficient mice as a model for human ARVC. These findings could provide for the opportunity to identify new diagnostic markers and therapeutic targets in patients with ARVC.

AB40-5: Endurance training aggravates arrhythmogenic right ventricular cardiomyopathy in heterozygous plakoglobin-deficient mice - evidence for functional conduction slowing in mice and men

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy affecting the right ventricle. ARVC is an important cause of sudden death in the young. Mutations in desmosomal proteins (plakophilin, desmoplakin, or plakoglobin) have been associated with ARVC. Whether reduced desmosomal protein function causes ARVC is not known. Ten months old, but not three or six months old, plakoglobin-deficient mice (plako -/+) developed increased right ventricular volumes, reduced right ventricular function, and spontaneous ventricular ectopy (all p<0.05 vs.