Myocardial citrullination in rheumatoid arthritis: a correlative histopathologic study

Rates of heart failure and diastolic dysfunction are increased in rheumatoid arthritis (RA) patients compared to non-RA controls independent of coronary artery disease [1], suggesting that myocardial remodeling occurs as part of the RA disease process. The phenotype of heart failure in RA differs from that of non-RA patients, characterized by fewer symptoms, lower blood pressure, and higher ejection fraction at presentation [2], suggesting that the pathophysiologic mechanisms underlying the progression to heart failure in RA may differ from the general population. Reports of increased myocarditis and vasculitis in autopsied RA hearts compared to controls date back more than five decades [3, 4]. However, the myocardium as a potential autoimmune target in rheumatoid arthritis has received little direct investigation since that time.

Recently, we reported an association of a higher concentration of serum anti-cyclic citrullinated peptide (anti-CCP) antibodies with lower myocardial mass and smaller left ventricular chamber volumes in RA patients without known cardiovascular disease [5]; raising the possibility that RA-specific autoimmunity against citrullinated proteins might mediate changes to myocardial morphology that, in turn, may affect myocardial function. Citrullination, the post-translational modification of arginine to citrulline catalyzed by a set of peptidyl-arginine deiminase enzymes (PADs), is abundant in the rheumatoid synovium but not restricted to RA [6]. Citrullinated proteins have been identified in the affected tissues of a number of inflammatory conditions, such as muscle in myositis patients, myelin sheaths in multiple sclerosis, and intestinal mucosa in inflammatory bowel disease [7] and in non-pathologic tissues with homeostatic functions, such as keratinized epithelium.

What are the potential links between circulating anti-citrullinated peptide antibodies (ACPA) and the myocardium? Many of the protein targets that undergo citrullination in rheumatoid synovium (that is, vimentin, enolase, fibronectin) and are targets for ACPA in RA [8] are also present in the myocardium [9]. However, it is unknown whether citrullination of myocardial proteins is present in RA. For this investigation, we explored the presence, abundance, and localization of citrullination in archived myocardial samples from RA patients compared to a variety of rheumatic and non-rheumatic disease control groups. Further, we explored co-localization of citrullination with histologic features and the presence of human PAD isotypes. We hypothesized that myocardial citrullination would be unique to, or more abundant in, RA compared to other conditions, and that myocardial regions demonstrating citrullination would co-localize with evidence of tissue damage (for example, myocarditis, fibrosis) and human PAD expression.

To the best of our knowledge, this is the first report of the presence of myocardial citrullination in any disease state, and the first histologic description of staining patterns of all known human PAD isotypes in myocardial tissues. These data provide evidence that: (1) citrullinated proteins are present in the myocardial interstitium of diverse disease states, but significantly increased in RA; (2) demographics, postmortem interval, and the presence or extent of reported coronary atherosclerosis did not account for higher citrullination in RA; and (3) a variety of PADs are present in the myocardium, but they do not co-localize with interstitial citrullination. Our finding that citrullination was indeed present in the myocardium extends the list of tissues known to undergo this form of post-translational modification. Although we did not identify specific citrullinated proteins in this investigation, the localization of citrullination to the interstitium may provide clues as to likely candidates. Proteins found in the interstitium are primarily those of extracellular matrix (ECM) and proteins that interact with ECM (for example, collagens, fibronectin, laminin) [11]; however, while these are the most common interstitial proteins, the list of additional candidate proteins remains extensive. Our finding that age was more strongly associated with citrullination in RA compared to controls could suggest that citrullination is an expected aging-related phenomenon accelerated in RA by processes that remain to be elucidated, perhaps related to accelerated apoptosis or autophagic death of cardiomyocytes or cardiac leukocytes. Neither myocarditis nor myocyte necrosis were frequent among RA samples, suggesting that fulminant processes such as these may not be the triggers for citrullination. Moreover, the lack of citrullination above control levels among fatal myocarditis patients further supports a lack of association. Another interesting finding was higher citrullination scores among RA samples with evidence of interstitial fibrosis. The association suggests that the two processes may be linked; however, whether interstitial fibrosis and citrullination are causally linked, or merely epiphenomena, is not evident from our study.

There is precedent for other mechanisms of post-translational modification of myocardial proteins leading to changes in cardiac structure and function. Phosphorylation, oxidation, and glycation of sarcomeric proteins cause morphologic changes to proteins that lead to increased protein cross-linking, resulting in increased ventricular stiffness [12]. Beyond direct structural effects related to altered protein-protein interaction, accumulation of modified proteins has been shown to mediate myocardial fibrosis via modulation of the expression of proteases and transcription factors in myocardial fibroblasts [13]. It is unknown whether citrullination of myocardial proteins leads to altered interaction of myocardial proteins or cellular processes leading to interstitial fibrosis similar to other post-translational modifications; however, our findings warrant additional study into possible mechanisms.

Although citrullination appears to be a generalized process, autoantibodies targeting citrullinated proteins are relatively specific for RA and, although occasionally observed in other autoimmune conditions, are uncommonly observed in healthy individuals [14]. Thus, RA patients may be uniquely at risk for autoreactivity against proteins that, upon citrullination, function as neo-epitopes for anti-citrulline autoantibodies. RA patients are known to exhibit a broader repertoire of reactivity against citrullinated peptides compared to non-RA patients with ACPA antibodies [8], a process potentiated by epitope spreading [15]. Although it is not known from our study whether myocardial citrullinated proteins are immune targets for circulating autoantibodies, or whether autoimmunity against citrullinated proteins mediates phenotypic modifications to cardiac structure or function, this is a goal of ongoing investigation.

The cellular localization of PAD isotypes within the myocardium has not been previously described. While PADs 1 and 3 are primarily regarded as epidermal in origin, mRNA for PAD1 has been identified in muscle tissue [16]. In addition, our finding of PADs 2 and 4 primarily in leukocytes is expected from previous investigation [17]. Somewhat unexpected was the observation of a clear separation between the locations of citrullination and PADs in the myocardium in all of the disease groups, a finding that warrants further study.

Some notable study limitations deserve acknowledgment. Most notably, samples derived from autopsy may not represent processes active in life, findings may be influenced by cause of death, samples tend to be derived from older individuals and thus may misrepresent natural history over the course of disease, and histopathology may be affected by the interval between death and sample collection (that is, PMI). Because of slow progression and infrequency of fulminant symptoms, the diagnosis and management of myocardial dysfunction in RA patients rarely requires endomyocardial biopsy, limiting the spectrum of myocardial samples available. Even so, samples collected for clinical diagnostics could be biased by the factors necessitating biopsy (that is, skewing toward patients with extreme findings). Regarding the potential for PMI to confound our findings, we did not observe an influence of PMI on anti-citrulline detection, suggesting that citrullination is stable post mortem. Another limitation was the lack of clinical data, particularly for RA features and treatments. While richer clinical data would be helpful in exploring factors linked to citrullination within the RA group, notably the presence or absence of ACPA, the absence of the clinical characteristics does not impugn the differences detected comparing RA vs. non-RA disease states. As with any IHC-based study, antibody titer can affect where staining is observed. We used a low titer for our anti-modified citrulline antibody and saw robust staining in the extracellular matrix. A 10-fold higher titer of antibody did show myocardial staining, but it was difficult to discern this from background staining indicating that myocyte citrullination needs to be confirmed by a separate method. Finally, a strength of the investigation was the development a priori of standardized data abstraction tools and criteria for each disease group, with samples analyzed only for cases identified as definite or highly probable.

In summary, our novel detection of citrullinated proteins within the interstitium of the myocardium may have implications for cardiac physiology, both for RA patients and the general population. More extensive citrullination linked to the RA disease state, and the more robust association of age and interstitial fibrosis observed in RA compared to non-RA groups, may contribute to higher rates of myocardial dysfunction and overt heart failure in RA that deserves additional investigation.