Quantitative Immunohistochemistry of Desmosomal Proteins (Plakoglobin, Desmoplakin and Plakophilin), Connexin-43, and N-cadherin in Arrhythmogenic Cardiomyopathy: An Autopsy Study

Abstract

Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder related to mutations in desmosomal proteins. The current study tests the hypothesis that immunohistochemical staining for desmosomal proteins is of diagnostic utility by studying autopsy-confirmed cases of ARVC.

Methods and results: We studied 23 hearts from patients dying suddenly with ARVC. Control subject tissues were 21 hearts from people dying from non-cardiac causes (n=15), dilated cardiomyopathy (n=3) and coronary artery disease (n=3). Areas free of fibrofatty change or scarring were assessed on 50 sections from ARVC (24 left ventricle, 26 right ventricle) and 28 sections from controls. Immunohistochemical stains against plakoglobin, plakophilin, desmoplakin, connexin-43, and N-cadherin were applied and area expression analyzed by computerized morphometry. Desmin was stained as a control for fixation and similarly analyzed. The mean area of desmin expression was similar in controls and ARVC (86% vs. 85%, p=0.6). Plakoglobin expression was 4.9% ± 0.3% in controls, vs. 4.6% ± 0.3% in ARVC (p=0.3). Plakophilin staining was 4.8% ± 0.3% in controls vs. 4.4% ± 03% in ARVC (p=0.3). Desmoplakin staining was 3.4% in controls vs. 3.2 ± 0.2% in ARVC (p=0.6). There were no significant differences when staining was compared between right and left ventricles (all p > 0.1). For non-desmosomal proteins, the mean area of connexin-43 staining showed no significant difference by presence of disease.

Conclusions: The small and insignificant decrease in junction protein expression in ARVC suggests that immunohistochemistry is not a useful tool for the diagnosis.

Keywords: ARVC; arrhytmogenic cardiomyopaty; autopsy.; sudden death.

Fig. (1)

ARVC, fibrofatty change. A: Section of left ventricle showing areas of fatty change with fibrosis surrounding individual myocytes. B: A section of left ventricle from a different patient, with areas of fat, randomly distribution in areas of scar and myocytes. C: A section of right ventricle single myocytes interspersed with areas of scarring and fatty change. D: Fibrofatty change in the right ventricle. Note fibrosis and fat, with collagen surrounding individual myocytes near the endocardial surface.

Fig. (2)

A. Immunohistochemical stain for gamma-catenin in a patient with ARVC. A digitized section with intercalated discs staining with brown diaminobenzidine counterstain. B. Staining after digital subtraction colorizing brown target as yellow, for computerized assessment of percentage of staining. In this case yellow percentage of myocyte (cyan) area was rendered as 3.97%.

Fig. (3)

Immunohistochemical staining, ARVC patients, digitized unmixed composite images, prior to color reassignment. A. Plakoglobin (antibody JUP). There is normal intensity of staining, similar to controls. B Plakoglobin (antibody JUP, higher magnification), showing normal staining intensity. B. Plakoglobin (antibody gamma catenin), showing normal intensity of staining. D. Plakophilin, showing normal intensity of staining. E. Desmoplakin, showing normal intensity of staining. F. N-cadherin, showing normal intensity of staining.

Fig. (4)

Immunohistochemical staining, control patients, digitized unmixed composite images, prior to color reassignment. A. Plakoglobin (antibody JUP) showing normal intensity of staining. B Plakoglobin (antibody JUP, higher magnification) showing normal intensity of staining. B. Plakoglobin (antibody gamma catenin) showing normal intensity of staining. D. Plakophilin. showing normal intensity of staining E. Desmoplakin showing normal intensity of staining. F. N-cadherin showing normal intensity of staining.