Local inflammatory reaction leads to cardiomyocyte necrosis with subsequent redistribution of gap junctions and dissociation of tight junctions. Among the many tight junction proteins, the greatest attention is given to coxsackie-adenovirus receptor (CAR). CAR participation in the pathogenesis of inflammatory diseases of the myocardium suggests that regulation of its expression depend on the degree of inflammation. CAR overexpression is known to maintain the inflammation in myocarditis . Whether the increased expression of this receptor on cardiomyocytes, is a result of an inflammatory condition of the myocardium, or merely a pathophysiologic finding contributing to the inflammatory process and myocardial remodeling, remains unclear. To address this question, we evaluated the intensity of the expression of CAR on the cardiomyocytes in relation to the degree of cellular infiltration of the myocardium in patients with DCM. Our data showed heterogeneous morphological changes in EMB in patients with similar clinical (symptoms of heart failure) and instrumental data (heart chamber dilatation and reduced left ventricular ejection fraction). Experimental models showed differntial CAR expression in different disease stages with maximum level appearing in the necrotic phase. CAR expression can be found in the cardiomyocytes not only in the inflammatory lesions but also in the intact area . Our study shows similar finding without CAR expression in the necrotic stage. However, similar to Kaur T. et al. who had shown no direct relationship between high CAR expression and severity of myocarditis in the autopsy material of DCM hearts, we failed to identify such relationship in our investigation.
Recognizing the important role of CAR in the pathogenesis of inflammatory DCM led to direct investigation of the factors influencing its expression. It is known that the expression of CAR is enhanced by increasing concentration of TNFα that can be seen in both the animal models of CVB3 induced myocarditis  and in cultured oncological cell . At the same time TGF-β1 was shown to block the TNFα influence and return the CAR concentration on membranes to baseline . This observation prompted us to examine serum cytokines. Our study revealed a relationship between myocardial and platelet CAR and high level of serum IL-6. Analysis of the data also indicates that CAR overexpression is typical for patients with severe heart failure. Taking into account the fact that the high concentration of IL-6 has cardiodepressive effect, leads to an increased collagen volume fraction, and consequently, myocardial stiffness , we can assume that increased IL-6 level enhances functional role of CAR as an intercellular tight junction protein. It is well known that in DCM membrane of intercalated disc are curved with a concomitant increase in the expression of adhesive contact, which may lead to a loss of flexibility and high rigidity in the sites of intercellular contacts . Besides changes on the tissue level changes of the cardiomyocytes’ structure contribute to the reduction of the heart contractility. The main event of the pathological remodeling of cardiomyocytes is a disruption of intercellular contacts. Pathological conditions lead to a significant decrease in the expression of other type of cell-cell contacts - gap junctions, which normally carry out electromechanical coupling between adjacent cardiomyocytes . We can propose that diffuse reduction in myocardial contraction of the left ventricle in patients with DCM can be determined by replacement of gap junction proteins, connexins, to proteins of tight junctions. It is likely that the observed CAR overexpression may reflect an attempt to restore intercellular contacts in the affected myocardium with progressive dilatation. And perhaps the purpose of tight junctions proteins, CAR for example, is to distinguish the intact cells from necrotic lysis products. A strong expression of the CAR protein can be observed in the intercalated discs and sarcolemma not only in the end stage human dilated cardiomyopathy (DCM), an up-regulation of CAR mRNA was revealed in ischemic cardiomyopathy (ICM), in myocardium of patients with valve-failure associated heart disease and in animal models of myocardial infarction . Thus, our results and results of other studies suggest a role for CAR in myocardium remodeling during pathological conditions regardless of its etiology.
The fact that peripheral proinflammatory cytokine – IL6 – is related to CAR expression on the membrane of platelets, blood cells, which are capable of immediately responding to systemic inflammation reminds us about their complex role in virus infection . Viral infection even that transient is considered to be the etiological factor of myocarditis and can induce platelet activation. Platelet activation can lead to elimination of virus laden platelets but also to the clearance of virus particles through release their contents from α-granules including high amount of CXCL4 which in turn up-regulates coagulation and leukocyte recruitment . Previous studies have confirmed platelet changes in patients with idiopathic DCM . It is likely that these changes contribute to the impairment of microcirculation, and thus to exacerbating of the disease. CAR expression on peripheral blood cells in patients with DCM was first described by Liu Q. et al. . They showed -in 50 patients with DCM-, CAR expression on leukocytes and CVB3 persistence inside them together. This is contradictory to our results which demonstrated that, CAR expression and persistence of Coxsackie virus in myocardium were not related. In our study 39.4% EMB samples were parvovirus B19-posititve. Virus persistence was not related to histological signs of myocarditis. Our data is fully consistent with the data of Marburg registry which showed that herpes virus type 6 and parvovirus B19 are typical for EMB samples in European population, while the coxsackie virus is a rare finding .
The notion that viruses can persist in peripheral blood together with platelet ability to internalize virus particles led us to identify -in a pilot study- 6 patients (30%) with mature CMV and HSV 1–2 virus particles inside platelets. Their persistence in platelets was not related to virus persistence in myocardium. It remains unclear whether these viruses are related to pathogenesis of inflammatory heart diseases or is just a bystander maintaining the systemic inflammatory process. Nevertheless, the fact that platelets can internalize viruses during cardiovascular pathology corresponds well with the previous in vitro and in vivo studies [16–20, 34].
Our data revealed that platelets of DCM patients are activated as evidenced by their increased aggregation. In most patients an increased level of spontaneous and low dose ADP-induced aggregation were observed. This indicates the changes in the platelets’ functional characteristics, and increased activity which enable them to form small aggregates (from 3 to 100 cells). The persistence of microaggregates, leukocyte- and erythrocyte-platelet aggregates (LPA, EPA) was confirmed by scanning electron microscopy. These findings correspond with the observation that in patients, CAR is expressed predominantly at the sites of intercellular communications in the small aggregates. One can suspect that platelet activation upon the inflammatory conditions regardless of its inducer lead to microaggregate, LPA and EPA formation, and CAR is one of the major proteins involved in this process. It seems that CAR is involved in the special ‘docking membrane’ formation resembling the intercalated disc in myocardium which can enable intercellular communication and signal transduction.
In summary, this study showed some novel key findings. First, CAR expression in myocardium is not related to the severity of inflammation or persistent virus infection but likely involved in the remodeling process during the pathological inflammatory conditions. Moreover, CAR overexpression both in myocardium and platelets is associated with systemic inflammation as evidenced by increased level of IL-6 in peripheral blood. Second, platelets of DCM patients are activated as evidenced by increased aggregation and microagregates; LPA and EPA. CAR is localized predominantly in such aggregates in the sites of intercellular contact forming the ‘docking membrane resembling the intercalated disc in myocardium. Third, 30% of assessed DCM patients revealed platelets which carried the internalized CMV and HSV-1 particles. Virus persistence inside platelets doesn’t reflect virus persistence in myocardium in DCM patients and may not be the etiological factor of inflammatory heart disease but rather a bystander in the systemic inflammatory process.
We conclude that tight junction protein CAR may be serving as docking pin forming a new type of contact structures whiсh might be responsible for signaling between neighboring cells during inflammation or other pathological conditions. Future investigation should examine further the physiological role of CAR expression in myocardium and its interaction with other types of intercellular connections especially gap junctions.