Altered desmosomal proteins in granulomatous myocarditis and potential pathogenic links to arrhythmogenic right ventricular cardiomyopathy

Abstract

Background: Immunoreactive signal for the desmosomal protein plakoglobin (γ-catenin) is reduced at cardiac intercalated disks in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), a highly arrhythmogenic condition caused by mutations in genes encoding desmosomal proteins. Previously, we observed a false-positive case in which plakoglobin signal was reduced in a patient initially believed to have ARVC but who actually had cardiac sarcoidosis. Sarcoidosis can masquerade clinically as ARVC but has not been previously associated with altered desmosomal proteins.

Methods and results: We observed marked reduction in immunoreactive signal for plakoglobin at cardiac myocyte junctions in patients with sarcoidosis and giant cell myocarditis, both highly arrhythmogenic forms of myocarditis associated with granulomatous inflammation. In contrast, plakoglobin signal was not depressed in lymphocytic (nongranulomatous) myocarditis. To determine whether cytokines might promote dislocation of plakoglobin from desmosomes, we incubated cultures of neonatal rat ventricular myocytes with selected inflammatory mediators. Brief exposure to low concentrations of interleukin (IL)-17, tumor necrosis factor-α (TNF-α), and IL-6 (cytokines implicated in granulomatous myocarditis) caused translocation of plakoglobin from cell-cell junctions to intracellular sites, whereas other potent cytokines implicated in nongranulomatous myocarditis had no effect, even at much higher concentrations. We also observed myocardial expression of IL-17 and TNF-α and elevated levels of serum inflammatory mediators, including IL-6R, IL-8, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1β, in patients with ARVC (all P<0.0001 compared with controls).

Conclusions: The results suggest novel disease mechanisms involving desmosomal proteins in granulomatous myocarditis and implicate cytokines, perhaps derived in part from the myocardium, in disruption of desmosomal proteins and arrhythmogenesis in ARVC.

Conflict of interest statement

Conflict of Interest Disclosures: None

Figures

Figure 1

A. Microscopic appearance of the myocardium from a patient with cardiac sarcoidosis showing fibrosis and multinucleated giant cells within granulomatous lesions (Masson’s trichrome stain, x20). B. Normal appearing myocardium from the same patient showing no apparent inflammatory or degenerative changes (Masson’s trichrome stain, x20). C. Representative confocal immunofluorescence images of control myocardium and myocardium from a patient with cardiac sarcoidosis clinically masquerading as ARVC and another patient with pathologically documented cardiac sarcoidosis. Specific immunoreactive signal for plakoglobin was significantly depressed in both cases compared to controls as was signal for the major gap junction protein, Cx43. Expression of other desmosomal proteins including desmoplakin and plakophilin-2 varied but signal for the non-desmosomal adhesion protein N-cadherin was always present and indistinguishable from controls.

Figure 2

A. Microscopic appearance of the myocardium from a patient with giant cell myocarditis showing severe destruction of cardiac myocytes (hematoxylin/eosin stain, x20). B. Normal appearing myocardium from the same patient showing no apparent inflammatory or degenerative changes (hematoxylin/eosin stain, x20). C. Representative confocal immunofluorescence images of control myocardium and myocardium from two patients with giant cell myocarditis (GCM). Specific immunoreactive signal for plakoglobin was significantly depressed in both cases compared to controls as was signal for the major gap junction protein, Cx43. Expression of other desmosomal proteins including desmoplakin and plakophilin-2 varied but signal for the non-desmosomal adhesion protein N-cadherin was present in all GCM cases analyzed and indistinguishable from controls.

Figure 3

A. Microscopic appearance of myocardium from a patient with lymphocytic myocarditis of viral etiology (influenza A) showing inflammatory infiltrates and myocyte necrosis (hematoxylin/eosin stain, x10). B. Normal appearing myocardium from the same patient showing no inflammatory infiltrate or degenerative changes (hematoxylin/eosin stain, x20). C. Representative confocal immunofluorescence images of control myocardium and myocardium from a patient with lymphocytic myocarditis. Specific immunoreactive signals for N-cadherin, plakoglobin, desmoplakin and plakophilin-2 were indistinguishable from controls. Signal for Cx43 at cell-cell junctions appeared diminished compared to controls.

Figure 4

A. Representative confocal immunofluorescence images showing the distribution of junctional plakoglobin in cardiac myocytes incubated with varying concentrations of selected cytokines. IL-17, TNFα and IL-6 caused obvious loss of plakoglobin signal even at the lowest concentration of 12.5ng/ml of medium while other cytokines including IL-4, IL-9, IL-12 and interferonγ (IFγ) had no effect even at a concentration of 400ng/ml medium. B. Representative immunoblots showing no apparent change in total cellular content of plakoglobin in myocytes incubated with varying concentrations of IL-17, TNFα and IL-6. Gluceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control.

Figure 5

Representative immunoperoxidase staining for IL-17, TNFα and IL-6 expression in myocardial samples from ARVC patients. A human breast carcinoma sample and myocardial samples from patients with sarcoidosis and giant cell myocarditis were used as positive controls. Myocardium obtained at autopsy from an individual with no history or pathological evidence of heart disease was used as a negative controls. The intensity of immunoreactive signal indicated by the brown reaction product was increased in ARVC myocardium compared with control myocardium for IL-17 and TNFα, but not IL-6.

Figure 6

Graphs showing the relative proportion of individuals within various concentration ranges for serum cytokines in which there were significant differences between ARVC patients and healthy controls. IL6R, interleukin-6 receptor; MIP1β, macrophage inflammatory protein 1beta; MCP1, monocyte chemoattractant protein-1; IL-8, interleukin-8; TNFαR1, tumor necrosis factor-α receptor type 1; TNFαR2, tumor necrosis factor-α receptor type 2.