Disorganization of intercalated discs in dilated cardiomyopathy

Abstract

Dilated cardiomyopathy (DCM) is a primary myocardial disease, the pathology of which is left ventricular or biventricular dilation and impaired myocardial contractility. The clinical and pathological diagnosis of DCM is difficult, and other cardiac diseases must be ruled out. Several studies have reported pathological findings that are characteristic of DCM, including cardiomyocyte atrophy, nuclear pleomorphism, and interstitial fibrosis, but none of these findings are DCM-specific. In this study, we examined the morphological differences in the intercalated discs (ICDs) between three groups of patients, a DCM group, a chronic heart failure group, and a control group. A total of 22 autopsy cases, including five DCM cases, nine CHF cases and eight control cases, were retrieved from the archives of the Department of Pathology at Akita University, Japan. The morphological differences were examined using multiple methods: macroscopic examination, light microscopy, immunohistochemistry, electron microscopy, and gene expression analyses. We observed disorganized ICDs, clearly illustrated by N-cadherin immunostaining in the DCM group. "Reduction of N-cadherin immunostaining intensity" and "ICD scattering" was DCM-specific. The results suggest that disorganized ICDs contribute to the development of DCM, and that N-cadherin immunostaining is useful for determining the presence of disorganized ICDs and for the pathological diagnosis of DCM.

Figure 1

Pathological and immunohistochemical findings in dilated cardiomyopathy. (A) Macroscopically, we observed severe dilation of the bilateral ventricles. LV wall thickness in the DCM group was relatively uniform, but the wall was thinner in DCM than in the other two groups. (B) ICDs were partially but not clearly observed, and cardiomyocyte units were unclear. (N-cadherin immunostaining; scale bar, 20 µm; original magnification, × 400). (C) Characteristic findings of DCM included cardiomyocyte atrophy, nuclear pleomorphism, and interstitial fibrosis (H–E staining; scale bar, 20 µm; original magnification, × 400). Immunohistochemistry revealed that N-cadherin immunostaining was lower in the DCM group than in the CHF and control groups (N-cadherin immunostaining; scale bar, 5 µm; original magnification, × 1000). ICD width was approximately 2–8 sarcomeres and was significantly elongated in the long-axis direction of the cardiomyocytes. The ICDs were scattered, had a stepwise shape, and were highly curved.

Figure 2

Pathological and immunohistochemical findings in control cases. (A) Macroscopically, we observed no dilation of the bilateral ventricles. The LV wall had a uniform thickness. (B) ICDs could be clearly observed, and cardiomyocyte units were clear. (N-cadherin immunostaining; scale bar, 20 µm; original magnification, × 400). (C) Histologically, ICDs were observed between cardiomyocytes (H–E staining; scale bar, 20 µm; original magnification, × 400). Each cardiomyocyte could be clearly distinguished. Immunohistochemistry for N-cadherin revealed positive staining at ICDs, and ICDs were thin and flat. (N-cadherin immunostaining; scale bar, 5 µm; original magnification, × 1000). ICD width was within two sarcomeres.

Figure 3

Pathological and immunohistochemical findings in cases of chronic heart failure. (A) Macroscopically, we observed mild-to-moderate dilation of the bilateral ventricles. The LV wall was thinner than in the control group. Depending on the cause of CHF, such as acute myocardial infarction, thickness was sometimes uneven. (B) ICDs could be seen relatively clearly, and the units of cardiomyocytes were clear. (N-cadherin immunostaining; scale bar, 20 µm; original magnification, × 400). The width of the ICDs was slightly wider in the CHF group than in the control group. (C) Histologically, ICDs were observed between cardiomyocytes, but their structures were somewhat irregular and hard to see (H-E staining; scale bar, 20 µm; original magnification, × 400). Immunohistochemistry for N-cadherin revealed that the width of the ICDs was approximately two to six sarcomeres, and was wider than in the control group (N-cadherin immunostaining; scale bar, 5 µm; original magnification, × 1000). The ICDs had a stepwise appearance and were crooked.

Figure 4

Transmission electron microscopy findings of intercalated discs in each group. (A) In the DCM group, ICDs were deteriorated and disorganized, the interdigitation of ICDs was disrupted throughout the tissue, and the junctions between cardiomyocytes and ICDs were obscured. Sarcomeres were arranged in a complex manner (scale bar, 1 μm; original magnification, × 6000). (B) In the control group, ICD ultrastructure was preserved. The finger-like folds of ICDs, called interdigitation, repeated at regular intervals (scale bar, 1 μm; original magnification, × 6000). (C) In the CHF group, ICDs were wider than in the control group, about two sarcomere widths. However, ICD ultrastructure as preserved. Interdigitation of ICDs was retained (scale bar, 1 μm; original magnification, × 6000).

Figure 5

Cardiomyocyte length, ICD width, and ICD scattering in each group. (A) Morphology of cardiomyocytes and ICDs in each group was observed in N-cadherin–stained specimens. Cardiomyocyte length (a), ICD width (b) and ICD scattering (c) were measured. (B) Cardiomyocyte length was significantly greater in the DCM group than in the control groups (a). ICD width was significantly greater in the DCM group than in the CHF and control groups (b), and differed significantly between the CHF group and the control group. The variation in the CHF cases was large. ICDs were more scattered in the DCM group than in the CHF and control groups (c). Statistical significance of differences between groups was determined using the Mann–Whitney U test: *P < 0.05.

Figure 6

Expression of genes associated with intercalated discs, and immunohistochemical staining of vinculin. (A) Expression of CDH2, CTNNB1, DSC2, DSG2, GJA1, TRPV2, and VCL genes in the LV samples from the three groups was evaluated by TaqMan quantitative real-time PCR. The gene expressions of ICD-associated genes, such as CDH2, CTNNB1, DSC2, DSG2, GJA1, TRPV2, and VCL, did not differ significantly between the DCM group and the CHF and control groups. The statistical significance of differences between groups was determined by the Mann–Whitney U test. (B) In the positive control group, immunohistochemistry revealed positive staining for vinculin in the ICD, myocardial cell membrane, and sarcomere. Immunostaining of vinculin was weaker than that of N-cadherin, and the ICD was hard to recognize. The ICD was lightly stained (vinculin immunostaining; scale bar, 5 µm; original magnification, × 1000). (C) As in the control group, the ICD stained lightly for vinculin. The myocardial cell membrane and sarcomere were also stained (vinculin immunostaining; scale bar, 5 µm; original magnification, × 1000). (D) In the DCM group, vinculin immunostaining was even weaker than in the CHF group, and ICDs were difficult to recognize. ICDs were deteriorated and disorganized (vinculin immunostaining; scale bar, 5 µm; original magnification, × 1000).