In this study, we report 2 major new findings: 1) disruption of desmosomal proteins in patients with granulomatous myocarditis, and 2) local myocardial production of selected cytokines and alterations in the balance between circulating pro-inflammatory and anti-inflammatory cytokines in patients with ARVC. Both of these findings implicate new disease mechanisms and establish potential new links between ARVC and selected forms of myocarditis.
To our knowledge, this is the first evidence to implicate disruption of desmosomal proteins in 2 forms of granulomatous myocarditis. We also observed that brief exposure of cardiac myocytes in vitro to exogenous cytokines implicated in granulomatous inflammation can cause redistribution of plakoglobin from junctional to intracellular sites, thus providing a potential mechanistic link. While there has been no previous demonstration of altered desmosomes in granulomatous myocarditis, it has been shown that desmogleins are targeted by auto-antibodies in pemphigus and by an exfoliative toxin from S. aureus
in bullous impetigo and staphylococcal scalded skin syndrome.33
It is also known that pro-inflammatory cytokines can disrupt tight junctions in intestinal epithelial cells.34
It is plausible to speculate, therefore, that cytokines produced locally or systemically in granulomatous myocarditis could affect desmosomes in the heart and contribute to cardiac myocyte injury and functional derangements. Because plakoglobin (γ-catenin) can participate in Wnt signaling pathways,35
our observations raise the possibility that altered Wnt signaling might also play a role in granulomatous myocarditis. Extensive additional work will be required to explore these possibilities, but our observations open new avenues of investigation for future studies.
Remodeling of gap junctions appears to be a consistent feature in ARVC.2,36,37
In the present study, we observed loss of immunoreactive signal for Cx43, the major ventricular gap junction protein, in some but not all cases of sarcoidosis and giant cell myocarditis, and there was not a strong correlation between loss of Cx43 signal and clinical expression of arrhythmias in these patients. Gap junction remodeling occurs in many forms of heart disease38,39
and was also seen in some patients with lymphocytic myocarditis in which loss of plakoglobin signal was not observed. Thus, the specific role of gap junction remodeling in arrhythmogenesis in ARVC and myocarditis remains unknown and further work is required to prove a causal relationship.
Pathologists have long recognized inflammation as a common feature of ARVC29
but it has not been shown whether it plays a primary pathogenic role or develops as a secondary response to myocyte injury and degeneration. Furthermore, inflammatory mechanisms in heart disease do not depend solely on the accumulation of inflammatory cells within the myocardium. Cardiac myocytes themselves can produce inflammatory mediators24,27–29
and circulating levels of various cytokines have been correlated with the development of atrial fibrillation40
or adverse outcomes in patients with heart failure.30–32
In the present studies, we found that selected cytokines, implicated in granulomatous inflammation but not other cytokines, were capable of promoting rapid intracellular translocation of junctional plakoglobin in cultured neonatal rat ventricular myocytes. Thus, our observations suggest that inflammatory mediators may play a role in ARVC even in the absence of infiltrating inflammatory cells in the heart. While this simple “proof-of-principle” study is of potential interest and worth exploring in future studies, its relevance in terms of disease mechanisms in granulomatous myocarditis and/or ARVC remains undefined. However, we also observed immunoreactive signal for TNFα and IL-17 in ARVC myocardium, thus raising the possibility that local production of cytokines could play a role in redistribution of plakoglobin and contribute to myocyte injury. Myocardial production of TNFα and its receptors has been reported in human heart failure.27,41
We also observed significant elevations in selected pro-inflammatory factors and a significant reduction in the level of IL-1 receptor 2, an anti-inflammatory protein that acts as a decoy receptor to bind IL-1 but not lead to downstream signaling.42
A recent study has reported elevated levels of IL-1β, IL-6 and TNFα in 8 ARVC patients.43
This study used a commercially available ELISA assay that has far lower sensitivity than the assay used in the present study,20
and reported values of <10 pg/ml for all measurements. Taken together, however, this previous study and our observations indicate that patients with ARVC exhibit significant increases in circulating inflammatory mediators, perhaps reflecting an imbalance between pro-inflammatory and anti-inflammatory proteins. Neither study was sufficiently powered to correlate patterns of cytokine expression with disease severity or clinical outcomes. Furthermore, we do not mean to suggest that elevated cytokine levels necessarily carry any diagnostic significance. Indeed, increased circulating inflammatory mediators have been reported in a variety of heart diseases including sarcoidosis and lymphocytic myocarditis.44,45
However, our observations provide a foundation for future studies in which analysis of serum inflammatory biomarkers in ARVC can be assessed in risk-stratification and correlated with arrhythmias or other manifestations of disease. These observations also raise the possibility of anti-inflammatory therapy in ARVC.
A limitation in the present study is the relative lack of mechanistic insights that could be gained from studying formalin-fixed, paraffin-embedded tissue samples. Nevertheless, we discovered potentially important new links between ARVC and some forms of myocarditis. We have also provided new information implicating cytokines in ARVC and new evidence that the myocardium itself may be the source of some of these mediators. Thus, disease pathways activated by mutations in desmosomal proteins may stimulate expression and possible secretion of inflammatory mediators which, in turn, could promote myocyte injury and/or arrhythmogenesis. Perhaps the most important strength of our observations is that they come from the actual human disease. In turn, they will stimulate and inform experimental studies and prospective clinical trials to test hypotheses about disease mechanisms, patient risk-stratification and new therapeutic targets.