A novel dominant mutation in plakoglobin causes arrhythmogenic right ventricular cardiomyopathy

Abstract

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.

Figure 1.

Electropherograms showing wild-type (A) and mutant (B) sequences of exon 2 of the gene encoding plakoglobin. The position of the insertion is marked with an arrow. C, Pedigree of family with ARVC. Squares denote males; circles denote females. The gray symbol denotes a deceased individual who did not undergo clinical evaluation but who was presumed to have been affected. Affected individuals fulfilled the Task Force criteria for ARVC. The index patient is marked with an arrow.

Figure 2.

Microscopic appearance of the cardiac biopsy sample from the proband (by Masson’s trichrome stain).

Figure 3.

Confocal immunofluorescence microscopy analysis of control and proband left ventricular myocardium, showing the amount of immunoreactive signal for selected junctional proteins at intercalated disks.

Figure 4.

A, Coomassie-blue staining of GST-fused N-terminal (N), central (mid), and C-terminal (C) domains of HRC-BP and GST. B, In vitro protein-binding assay, which shows that wild-type plakoglobin does not bind any of the three HRC-BP fragments (lanes 1–3), whereas mutant plakoglobin binds all three fragments in vitro (lanes 4–6). C, Lysates of cell lines induced to express wild-type (lane 1) and mutant (lane 2) plakoglobin, shown for control purposes. Coimmunoprecipitation of wild-type PG and TAIP-2 (lane 3), mutant PG and TAIP-2 (lane 4), wild-type PG and V5 vector (lane 5), mutant PG and V5 vector (lane 6), His Max vector and TAIP-2 construct (lane 7), and His Max vector and V5 vector only (lane 8), showing that both wild-type and mutant plakoglobin bind TAIP-2 in vitro.

Figure 5.

A, Four noninduced cell lines (lanes 2, 4, 6, and 8) that express the endogenous plakoglobin only (82 kDa), compared with four induced cell lines (lanes 1, 3, 5, and 7) that express both endogenous plakoglobin (82 kDa) and the mutant form of plakoglobin, which migrates at ∼90 kDa. Signal for endogenous plakoglobin is faint in lanes 2, 3, 5, 7, and 8. B, Bands showing that ubiquitin is present in the cell line expressing the highest levels of the exogenous construct (from lane 1 in panel A), both in its free form (8 kDa) and at a form (∼90 kDa) consistent with its being covalently bound to mutant plakoglobin.

Figure 6.

Confocal microscopy showing the distribution of wild-type and mutant plakoglobin in transiently transfected HEK293 cells. The preparations were counterstained with DAPI to show nuclei.

Figure 7.

A, CellTiter 96 Cell Proliferation Assay over the course of 5 consecutive d. A single asterisk (*) indicates P=.014; a double asterisk (**) indicates P=.006. B, CaspACE assay. Specific caspase activity was measured in 10 individual wild-type (wt) and mutant (mut) cultures. An asterisk (*) indicates P=.038.

Figure 8.

Electron micrographs of HEK293 cells stably expressing wild-type or mutant plakoglobin. The arrows indicate adhesive junctions between adjacent cells.