Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial Fibrillation

It is well established that the consumption of fish is associated with lower rates of cardiovas‐ cular death (Albert CM,et al.,2002; Hu FB, et al.,2002). Dietary fish oil supplementation has been shown to reduce mortality in high-risk groups through a reduction in sudden cardiac death and ventricular tachyarrhythmia. It has been recently reported that atrial fibrillation (AF) is associated with inflammation and inflammatory cytokines, and n-3 polyunsaturated fatty acids (PUFAs) might be of anti-inflammatory effects. Whether PUFAs has some antiar‐ rhythmic effect and can be used in the treatment of AF is still unknown.


Review of the relationship between n-3 polyunsaturated fatty acids and the atrial fibrillation
It is well established that the consumption of fish is associated with lower rates of cardiovascular death Hu FB, et al.,2002). Dietary fish oil supplementation has been shown to reduce mortality in high-risk groups through a reduction in sudden cardiac death and ventricular tachyarrhythmia. It has been recently reported that atrial fibrillation (AF) is associated with inflammation and inflammatory cytokines, and n-3 polyunsaturated fatty acids (PUFAs) might be of anti-inflammatory effects. Whether PUFAs has some antiarrhythmic effect and can be used in the treatment of AF is still unknown.

Dietary n-3 PUFA supplementation attenuates the inducibility and maintenance of AF
We established canine sterile pericarditis model and evaluate the anti-inflammatory effect of PUFAs on AF(Zhong Zhang,et al.,2010). Twenty mongrel sex-matched adult dogs were randomly divided into two groups. In the n-3 PUFA group (n=10), oral administration ofeicosa-pentaenoic+docosahexaenoic acid (EPA+DHA), 2 g/day (Omacor, Solvay Pharmaceuticals GmbH, Hanover, Germany) was started 4 weeks before the baseline study, and was continued until the end of the study. The dogs in the control group (n=10) did not receive n-3 PU-FAs or plant oil for 4 weeks. We examined the plasma concentration of the CRP, IL-6, and TNF-α before the operation and on the second postoperative day in both groups. There were no significant differences in three biomarkers of inflammation between two groups before the operation, and these biomarkers were significantly increased in both groups on the second postoperative day. However, three proinflammatory cytokines were significantly lower in the PUFA group than in the control group respectively (CRP, 7.6±0.5 vs. 11.7± 1.3 mg/dl, Pb0.0001 Fig. 1; IL-6, 112.0±37.3 vs. 142.0±19.6 pg/ml, Pb0.0001 Fig. 2; TNF-α, 83.3±8.5 vs. 112.4±8.2 pg/ml, Pb0.0001 Fig. 3).

Figure 1.
Comparison of CRP levels between the control and PUFA groups before and after operation. Before the operation, there were no significant differences in CRP levels between two groups. On the second postoperative day, CRP was significantly increased in both groups; however, it was significantly lower in the PUFA group than in the control.
The main finding of this study is that EPA and DHA supplementation of the diet can decrease plasma concentration of the CRP, IL-6 and TNF-α in acute inflammation of canine sterile pericarditis, suggesting depression of inflammatory cytokines by n-3 FUFAs may involve in the anti-atrial fibrillation process. The results also showed that the PUFA group had a less AF inducibility and maintenance than the control group (Table1).
Thus we may reasonably conclude that Dietary n-3 PUFA supplementation attenuates the inducibility and maintenance of AF in the sterile pericarditis model by reducing the production of proinflammatory cytokines. Comparison of IL-6 levels between the control and PUFA groups before and after operation. Before operation, there were no significant differences in IL-6 levels between two groups. On the second postoperative day, IL-6 was significantly increased in both groups; however, it was significantly lower in the PUFA group than in the control. Comparison of TNF-α levels between the control and PUFA groups before and after operation. Before the operation, there were no significant differences in TNF-α levels between two groups. On the second postoperative day, TNF-α was significantly increased in both groups; however, it was significantly lower in PUFA group than in the control.
Effect of CD3+ T-Lymphocyte and n-3 Polyunsaturated Fatty Acids on the Diagnosis or Treatment of Atrial

Inflammation and post cardiac surgery AF
Although the pathophysiological mechanism underlying the genesis of post cardiac surgery AF has been the focus of many studies,it only remains partially understood. The inflammatory cascade and catecholamine surge associated with surgery have been thought of playing a prominent role in initiating AF after cardiac surgery. As a prototypic marker of inflammation, CRP has been the focus of many studies, which is driven by the proinflammatory cytokines interleukin (IL)-1, tumor necrosis factor (TNF)-α, and IL-6 (Bruins P et al.,1997) found that IL-6 rises initially and peaks at 6 h after cardiac surgery, and CRP levels increases and peaks on the second postoperative day of the cardiac surgery, with complement-CRP complexes levels peaking on the 2nd or 3rd postoperative day. The incidence of atrial arrhythmias similarly peaks on postoperative day 2 or 3. Other researchers have confirmed that IL-6 and CRP increased after cardiac surgery, with the incidence of atrial arrhythmias similarly peaking on the 2nd or 3rd postoperative day [13,14] Other studies correlated leukocytosis to an increased incidence in AF in postoperative cardiovascular patients [16,17](Abdelhadi RH,et al.,2004;Lamm G,et al., 2006), and found that a more pronounced increase in postoperative WBC count will independently predict development of postoperative AF. Moreover, atrial inflammation of cardiac surgery effects on the electrical properties of atrial tissue, and the degree of atrial inflammation was associated with a proportional increase in the inhomogeneity of atrial conduction and AF duration [18] ( Ishii Y, et al.,2005). Administration of anti-inflammatory drugs (dexamethasone or cortisone) significantly decreases the incidence of AF after cardiac surgery [19,20] ,1986). According to the multiple-wavelet hypothesis, atrial wavelength determines the number of wavelets, and the atrial wavelength is the product of AERP and the intra-atrial conduction velocity. So, the AERP and the intra-atrial conduction velocity have been thought to be important for the perpetuation of AF. In this canine sterile pericarditis model, we have evaluated CRP, IL-6 and TNF-α level on the baseline and on the 2nd postoperative day, and found that they all significantly increased in both groups. We simultaneously evaluated the role of inflammation on atrial electrophysiological properties, and found that inflammation can shorten AERPs and prolong intra-atrial CT in the canine sterile pericarditis model, which increased the inducibility and stability of AF. Our results are concordant with the previous results. Thus, in this model, elevated CRP, IL-6 and TNF-α were associated with sustained AF, suggesting that electrophysiological changes resulting from inflammation perpetuate AF.

Other potential mechanisms of antiarrhythmic action of n-3 PUFA administration
The current hypotheses of n-3 PUFAs in preventing AF are based on their inhibiting capacity of some ion channels. Previous studies have demonstrated that n-3 PUFAs have capacity to inhibit fast, voltage dependent sodium currents, L-type calcium currents, the Na /Ca2 exchanger, which might prevent delayed after-depolarizations and triggered activity, as well as their class III antiarrhythmic-like effect on Kv1.  Fifty paroxysmal AF patients and fifty-six persistent AF patients, underwent successful electrical cardioversion, were enrolled in this study. Percentage of CD69 and Human leukocyte antigen DR (HLA-DR) positive peripheral blood CD3 + T-lymphocyte, which indicates Tlymphocyte activation, were examined by flow cytometric analysis in the patients and fiftyone healthy controls. Patients groups had higher levels of CD69 and HLA-DR than healthy controls. During three-month follow-up, 37 patients had recurrence of AF (recurrence group) and 50 patients remained in sinus (sinus group).

The expression of CD69 and CD3+ T-lymphocytes in the diagnosis or therapy of AF
The results showed that Patients with AF groups had higher levels of CD69 and HLA-DR than healthy controls.  Figure 4)   Before we received the result,we conduct the baseline clinical Caracteristics of the Studied Population to ensure facticity of the results ( ACEI indicates angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor blocking agents; CCBs, calcium channel blockers; WBC, white blood cells; CRP, C-reactive protein; p, probability of significance (difference among three groups); and *, p<0.05 persistent AF vs paroxysmal AF.
In this study, we found that the respective expression of CD69 and HLA-DR on peripheral blood CD3 + T-lymphocytes in AF patients was significantly higher than control group, which might suggest that high expression of CD69 and HLA-DR was associated with AF. In the subsequent follow-up, we further found that the expression of CD69 and HLA-DR in sinus group at follow-up was significantly down-regulated compared with before cardioversion. However, the expression of CD69 and HLA-DR in recurrence group at follow-up was not significantly down-regulated. It might further support that the CD69 and HLA-DR levels were related with the state of AF.
As demonstrated by the present study, there was a link between high expression of CD69 and HLA-DR and AF. CD69, known as an early activation marker of lymphocytes, is a type II transmembrane glucoprotein and may enhance activation and proliferation/differentiation of T-lymphocytes. ( Generally, our study here further emphasize that activation of T cells is involved in AF. As we all know, T-lymphocytes are the main cells participate in cell-mediated immunity, which is one of the primary ways of human immune. It can be suppressed by many immunosuppressants such as Cyclosporine, Rapamycin, et al. If the activation of peripheral blood Tlymphocytes does participate in the pathogenesis of AF, maybe we can prevent recurrence of AF through suppressing the activation of peripheral blood T-lymphocytes. As for the underlying mechanisms of activated peripheral blood CD3 + T-lymphocytes participates in the progression of AF, we speculate the following three possibilities. Firstly, the activation of peripheral blood CD3 + T-lymphocytes might cause the upregulation of IL-6 and MCP-1, which could affect the contractility and electrical stability of myocytes inhomogeneously and induce fibroblast activation leading to deposits of extracellular matrix fibrosis. It is important to note that there are several limitations that need to be addressed regarding this study. Firstly, The results cannot be taken as evidence to support that CD69 and HLA-DR play a role in the pathogenesis of AF, it only indicate the possible association of CD69 and HLA-DR with AF. Secondly, how CD69 and HLA-DR contribute to the pathogenesis of AF and what is the underlying mechanism need to be further investigated. Thirdly, we did not study the influence of other immune activation-associated molecules (CD25, CD71, and CD122, et al) and co-stimulatory molecules (CD28, CTLA-4, CD80, CD86, et al) during the progression of AF.

Summary
The activation of peripheral blood CD3+ T-lymphocytes and immunologic inflammatory responses played a role in the pathogenesis of AF,and might be a diagnostic or therapeutic marker. Dietary n-3 PUFA supplementation attenuates the inducibility and maintenance of AF in the sterile pericarditis model by reducing the production of proinflammatory cytokines.

Author details
Qiang-Sun Zheng, Hong- Tao  Hong-Tao Wang and Zhong Zhang contributed equally in this work.