Characterizing the Molecular Pathology of Arrhythmogenic Cardiomyopathy in Patient Buccal Mucosa Cells

Abstract

Background: Analysis of myocardium has revealed mechanistic insights into arrhythmogenic cardiomyopathy but cardiac samples are difficult to obtain from probands and especially from family members. To identify a potential surrogate tissue, we characterized buccal mucosa cells.

Methods and results: Buccal cells from patients, mutation carriers, and controls were immunostained and analyzed in a blinded fashion. In additional studies, buccal cells were grown in vitro and incubated with SB216763. Immunoreactive signals for the desmosomal protein plakoglobin and the major cardiac gap junction protein Cx43 were markedly diminished in buccal mucosa cells from arrhythmogenic cardiomyopathy patients with known desmosomal mutations when compared with controls. Plakoglobin and Cx43 signals were also reduced in most family members who carried disease alleles but showed no evidence of heart disease. Signal for the desmosomal protein plakophilin-1 was reduced in buccal mucosa cells in patients with PKP2 mutations but not in those with mutations in other desmosomal genes. Signal for the desmosomal protein desmoplakin was reduced in buccal mucosa cells from patients with mutations in DSP, DSG2, or DSC2 but not in PKP2 or JUP. Abnormal protein distributions were reversed in cultured cells incubated with SB216763, a small molecule that rescues the disease phenotype in cardiac myocytes.

Conclusions: Buccal mucosa cells from arrhythmogenic cardiomyopathy patients exhibit changes in the distribution of cell junction proteins similar to those seen in the heart. These cells may prove useful in future studies of disease mechanisms and drug screens for effective therapies in arrhythmogenic cardiomyopathy.

Keywords: arrhythmogenic right ventricular cardiomyopathy; buccal mucosa cells; connexin43; desmosome cardiomyopathy; diagnosis; plakoglobin.

Figure 1

Representative images of normal buccal mucosa smears. Cells stained with hematoxylin and eosin (H&E) show typical squamous morphology with central nuclei and clearly delineated cell borders. Buccal mucosa cells immunostained with antibodies against desmosomal and gap junction proteins show strong immunoreactive signals for plakoglobin, desmoplakin, plakophilin-1 and Cx43 concentrated at the edges of the cells. No apparent signal is seen for plakophilin-2 which is expressed in the heart but not in epithelial tissues. Cell nuclei (blue) are stained with DAPI.

Figure 2

Representative images of buccal mucosa smears from a control, and ACM patients with mutations in PKP2 or DSP. Both patients show loss of junctional signal for plakoglobin and Cx43 compared to controls. Cells from the patient with a PKP2 mutation show loss of signal for plakophilin-1 but not desmoplakin, whereas cells from the patient with a DSP mutation show loss of signal for desmoplakin but not plakophilin-1. Cell nuclei (blue) are stained with DAPI.

Figure 3

Representative images of cultured buccal mucosa cells obtained from a control subject, an ACM patient and a clinically unaffected carrier of a PKP2 mutation, before (A) and after (B) exposure to SB216763. Cultured cells from both the patient and carrier showed loss of junctional signal for plakoglobin, accumulation of nuclear plakoglobin and loss of junctional Cx43. Incubation with SB216763 for 24 hours normalized plakoglobin and Cx43 signal distributions. Cell nuclei (blue) are stained with DAPI.