Increased serum high mobility group box 1 protein in patients with atrial fibrillation

Results HMGB1 level in persistent AF group (8.62 ± 2.18 ng/ml) was higher than that in control group (2.22 ± 0.63 ng/ml) and paroxysmal AF group (5.29 ± 1.43 ng/ml) (both P < 0.05). HMGB1 level in paroxysmal AF group was higher than that in control group ( P < 0.05). There was significantly positive correlation between HMGB1 and hs-CRP in AF patients ( n = 88, r = 0.629, P < 0.05). Conclusion The present study showed that serum HMGB1 level was markedly increased in paroxysmal and persistent AF patients. Keywords High mobility group box 1 protein Atrial fibrillation Inflammation Abbreviations AF atrial fibrillation hs-CRP high-sensitive C-reactive protein HMGB1 high mobility group box 1 protein LAD left atrial diameter LVEDD left ventricular end-diastolic dimension LVEF left ventricular ejection fraction BMI body mass index WBC white blood count ROS reactive oxygen species 1 Introduction AF is the most common sustained cardiac rhythm disturbance in clinical practice, increasing in prevalence with age. The estimated prevalence of AF is 0.4 to 1% in the general population, increasing with age to 8% in those older than 80 years [1,2] . In prospective studies, the incidence of AF increases from less than 0.1% per year in people younger than 40 years to over 1.5% per year among women and 2% among men older than 80 years [3] . Despite the extensive studies, the pathophysiological mechanisms underlying the genesis of AF remain unclear. More recently, the bulk of evidence suggests that inflammatory process is associated with development and recurrence of AF [4] . Many studies showed that several pro-inflammatory cytokines, such as hs-CRP, CRP or interleukin-6 (IL-6) were significantly increased in patients with AF and may be associated with greater risk of AF recurrence after electrical cardioversion and catheter ablation [4–6] . These results suggest that pro-inflammatory cytokines may play an important role in development and recurrence of AF and be a possible pathogenic link to AF. HMGB1, a nonchromosomal nuclear protein, could regulate gene transcription and maintain the nucleosome structure [7] . In 1999, Wang et al. [8] first demonstrated that HMGB1 functioned as a delayed mediator in inflammatory responses in sepsis and showed that the inhibition of HMGB1 confers significant protection against the lethal effects of endotoxin, indicating that the extracellular HMGB1 plays a important role in the pathogenesis of sepsis. Recent studies revealed that HMGB1 also functioned as a novel pro-inflammatory cytokine in cardiovascular diseases [9,10] . In addition, Salman et al. [11] showed that the incidence of paroxysmal AF is high in critically ill patients with sepsis (HMGB1 play the critical role in the pathogenesis of sepsis [8] ), suggesting that HMGB1 may contribute to the genesis of paroxysmal AF in sepsis patients. However, whether serum HMGB1 was increased in AF patients remain unknown. In the study, we aimed to investigate whether the serum HMGB1 level was increased in patients with AF. 2 Materials and methods 2.1 Study subjects This clinical protocol was approved by the institutional medical Ethics Committee and conducted according to the ethical guidelines outlined in the Declaration of Helsinki. A total of 88 consecutive AF patients were enrolled from Renmin Hospital of Wuhan University, P.R. China. Another 30 age- and sex-matched healthy people who agreed to participate in this study were considered as control group. Detailed medical history, physical examination, routine biochemical testing, 12-lead electrocardiograms were performed both in AF patients and control group. Valvular functions, left ventricular size (LVEDD) and functions (ejection fraction, EF), and LAD were evaluated by transthorasic echocardiography. The diameter of the left atrium was measured in parasternal long axis view. AF duration was determined by the patient's description of a well-defined and abrupt onset palpitation with subsequent electrocardiograms evidence of AF at the time of presentation. Exclusion criteria were as follows: coronary artery disease, valvular heart disease, LVEF less than 45%, other types arrhythmias, surgery or stroke within 6 months, a history of infection, chronic inflammatory, hepatic or malign disease, chronic renal failure, autoimmune diseases, abnormal thyroid function, imbalance of electrolytes, use of anti-inflammatory drugs such as corticoids, nonsteroidal anti-inflammatory drugs excluding aspirin. 2.2 AF classification AF lasting less than or equal to 7 days is defined as new onset. After two or more episodes, AF is considered recurrent. If the arrhythmia terminates spontaneously, recurrent AF is designated paroxysmal; when sustained beyond 7 days, it is termed persistent which may include permanent AF. First-detected AF may be either paroxysmal or persistent. Lone AF was defined as AF occurring in the absence of structural heart disease and hypertension [12] . 2.3 Sample collection and biochemical investigation Peripheral venous blood was drawn from the antecubital vein after a 12 h fasting period between 7:00 and 8:00 AM in the morning. Serum samples were aliquoted and stored at −70 °C until being used. All samples were thawed only once. Serum hs-CRP was measured with standard laboratory techniques on a Hitachi 912 Analyzer (Roche Diagnostics, Germany). Serum HMGB1 level was determined with a commercially available ELISA kit (HMGB1 ELISA kit II; Shino-Test Corporation, Tokyo, Japan) according to its protocol. 2.4 Statistical analysis Statistical analysis was performed with the SPSS 16.0 s (SPSS Inc., Chicago, IL, USA). Data was presented as mean ± SD or the percentage of incidence. Chi 2 test or Fisher's exact test was used to compare proportions. One-way ANOVA or Welch was used for comparisons among groups and the Student-Neuman-Keuls or Dunnett T3 was used for post-hoc multiple comparisons. Pearson correlation coefficient was used to assess the relationship between serum HMGB1 concentrations and other parameters. Statistical significance was defined as P < 0.05. 3 Results 3.1 Clinical characteristics of patients As shown in Table 1 , there were no significant differences in percentages of sex, smoking, drinking, hyperlipemia, hypertension, diabetes and mediations (except digoxin which may be used it for controlling ventricular rate), age, BMI, LVEDD, EF and WBC among three groups. However, significant differences in HMGB1 and hs-CRP were found (both P < 0.05). HMGB1 level in persistent AF group (8.62 ± 2.18 ng/ml) was higher than that in control group (2.22 ± 0.63 ng/ml) and paroxysmal AF group (5.29 ± 1.43 ng/ml) (both P < 0.05). HMGB1 level in paroxysmal AF group was higher than that in control group ( P < 0.05). Hs-CRP level in persistent AF group (4.20 ± 1.04 mg/ml) was higher than that in control group (0.98 ± 0.32 mg/ml) and paroxysmal AF group (2.11 ± 0.94 mg/ml) (both P < 0.05). Hs-CRP level in paroxysmal AF group was higher than that in control group ( P < 0.05). The LAD and AF duration in persistent AF group were larger or longer than those in control group and paroxysmal AF group (both P < 0.05). The AF duration in paroxysmal AF group was longer than that in control group ( P < 0.05). The LAD in paroxysmal AF group was larger than that in control group, but no statistical significance was found ( P > 0.05). 3.2 Association of HMGB1 levels of cardiovascular risk factors As shown in Table 2 , there was significant correlation between HMGB1 and hs-CRP in AF patients ( n = 88, r = 0.629, P < 0.05). There were significant correlations with age and LAD, but no correlations with BMI, LVEDD, EF, AF duration and WBC. 4 Discussion In the present study, we found that serum HMGB1 level was significantly increased in paroxysmal and persistent AF patients. Several studies demonstrated that HMGB1 is a necessary and sufficient mediator of lethal inflammation, including in cardiovascular diseases [7–10,13] . HMGB1 is passively released by necrotic and damaged cells, apoptotic cell or by activated innate immune cells (such as macrophages and monocytes), and functions as a pro-inflammatory cytokine [7] . Once released from necrotic cells, apoptotic cell and macrophages, HMGB1 functions as an inflammatory stimulus that upregulates IL-1, IL-6, TNF-α, and macrophage inflammatory proteins (MIP-1α and MIP-1β) [7,14,15] . In the study, we found that the serum level of HMGB1 was positively correlated with the serum level of hs-CRP which was significantly increased in AF patients and was an important predictor for successful cardioversion and maintenance of sinus rhythm after conversion in AF patients [5,16] . This is consistent with the previous observations [10,17] . Hu [10] and Yan et al. [17] demonstrated that HMGB1 expression was closely correlated with hs-CRP, tumor necrosis factor-α (TNF-α) and IL-6 in patients with coronary artery diseases. These findings indicated that there was a cross-talk between HMGB1 and other pro-inflammatory cytokines including CRP which was associated with greater risk of AF recurrence after successful electrical cardioversion [4] . Inoue et al. [18] showed that activated vascular smooth muscle cells were the source of HMGB1 in human advanced atherosclerotic lesions and HMGB1 could directly stimulate the production of CRP. Meanwhile, Kawahara et al. [19] demonstrated that CRP dose-dependently induced the production of HMGB1 through the p38MAPK pathway. HMGB1, in return, triggered the expression of inflammatory cytokines, indicating that this mechanism reinforces the inflammatory process [7,14,18,19] . These studies suggest that HMGB1 may be a critical pro-inflammatory cytokine in the pathogenesis of AF. Therefore, HMGB1 up-regulates in patients with AF and may be involved in the pathophysiological mechanisms as well as others inflammatory cytokines [4–6] . A growing body of evidence suggests that oxidative stress may cause atrial structural and electrical remodeling and play an important role in the pathogenesis of AF [5,6,20,21] . However, it may be involved in the release of pro-inflammatory cytokine - HMGB1. Tang et al. [22] indicated that hydrogen peroxide, one of ROS, could stimulate macrophages and monocytes to actively release HMGB1. Tsung et al. [23] further demonstrated that HMGB1 release from cultured hepatocytes was also found to be an active process regulated by ROS. These results suggest that there was a cross-talk between oxidative stress and pro-inflammatory cytokine - HMGB1, oxidative stress may reinforce the effect of inflammation on the pathogenesis of AF. This is consistent with current concepts in the pathogenesis of AF [5,6,20,21] . Thus, we postulate that the role of oxidative stress in the pathogenesis of AF may be due to the release of HMGB1 and HMGB1 may play a critical role in the pathogenesis of AF. 5 Limitation Overall, our study included only a small group of Chinese patients, and a future study with a large cohort will be needed. The precise mechanisms underlying our observations require future elucidation. In addition, there were lots of risk factors for AF, however, we only analyzed a few typical risk factors in this study. 6 Conclusions In the present study, serum HMGB1 level was markedly increased in paroxysmal and persistent AF patients. These suggested that HMGB1 may be associated with the development and recurrence of AF, and may possibly be involved in the pathogenesis of AF. It may provide a potential target for pharmacological interruption of AF. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement This study was supported by the Science Research Foundation of Wuhan University for MD candidate to Xiaorong Hu. References [1] C.D. Furberg B.M. Psaty T.A. Manolio Prevalence of atrial fibrillation in elderly subjects (the Cardiovascular Health Study) Am J Cardiol 74 1994 236 241 [2] A.S. Go E.M. Hylek K.A. Phillips Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the anticoagulation and risk factors in atrial fibrillation (ATRIA) study JAMA 285 2001 2370 2375 [3] B.M. Psaty T.A. Manolio L.H. Kuller Incidence of and risk factors for atrial fibrillation in older adults Circulation 96 1997 2455 2461 [4] T. Liu G. Li L. Li P. Korantzopoulos Association between C-reaction protein and recurrence of atrial fibrillation after successful electrial cardioversion J Am Coll Cardiol 49 2007 1642 1648 [5] D.I. Leftheriotis K.T. Fountoulaki P.G. Flevari The predictive value of inflammatory and oxidative markers following the successful cardioversion of persistent lone atrial fibrillation Int J Cardiol 135 2009 361 369 [6] J. Li J. Solus Q. Chen Role of inflammation and oxidative stress in atrial fibrillation Heart Rhythm 7 2010 438 444 [7] P. Scaffidi T. Misteli M.E. Bianchi Release of chromatin protein HMGB1 by necrotic cells triggers inflammation Nature 418 2002 191 195 [8] H. Wang O. Bloom M. Zhang HMG-1 as a late mediator of endotoxin lethality in mice Science 285 1999 248 251 [9] T. Kohno T. Anzai K. Naito Role of high-mobility group box 1 protein in post-infarction healing process and left ventricular remodelling Cardiovasc Res 81 2009 565 573 [10] X. Hu H. Jiang Q. Bai Increased serum HMGB1 is related to the severity of coronary artery stenosis Clin Chim Acta 406 2009 139 142 [11] S. Salman Bajwa O. Gajic Paroxysmal atrial fibrillation in critically ill patients with sepsis J Intensive Care Med 23 2008 178 183 [12] V. Fuster L.E. Ryden D.S. Cannom ACC/AHA/ESC 2006 guidelines for the management of patients with atrial fibrillation: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the European Society of Cardiology Committee for Practice Guidelines (Writing Committee to Revise the 2001 Guidelines for the Management of Patients With Atrial Fibrillation): developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society Circulation 114 2006 e257 e354 [13] S. Yamada I. Maruyama HMGB1, a novel inflammatory cytokine Clin Chim Acta 375 2007 36 42 [14] U. Andersson H. Wang K. Palmblad High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes J Exp Med 192 2000 565 570 [15] C.W. Bell W.W. Jiang C.F. Reich The extracellular release of HMGB1 during apoptotic cell death Am J Physiol Cell Physiol 291 2006 C1318 C1325 [16] E. Watanabe T. Arakawa T. Uchiyama I. Kodama H. Hishida High-sensitivity C-reactive protein is predictive of successful cardioversion for atrial fibrillation and maintenance of sinus rhythm after conversion Int J Cardiol 108 2006 346 353 [17] X.X. Yan L. Lu W.H. Peng Increased serum HMGB1 level is associated with coronary artery disease in nondiabetic and type 2 diabetic patients Atherosclerosis 205 2009 544 548 [18] K. Inoue K.I. Kawahara K. Krishna HMGB1 expression by activated vascular smooth muscle cells in advanced human atherosclerosis plaques Cardiovasc Pathol 16 2007 136 143 [19] K. Kawahara K.K. Biswas M. Unoshima C-reactive protein induces high mobility group box-1 protein release through activation of p38MAPK in macrophage RAW264.7 cells Cardiovasc Pathol 17 2008 129 138 [20] P. Korantzopoulos T.M. Kolettis D. Galaris The role of oxidative stress in the pathogenesis and perpetuation of atrial fibrillation Int J Cardiol 115 2007 135 143 [21] D.R. Van Wagoner Oxidative stress and inflammation in atrial fibrillation: role in pathogenesis and potential as a therapeutic target J Cardiovasc Pharmacol 52 2008 306 313 [22] D. Tang Y. Shi R. Kang Hydrogen peroxide stimulates macrophages and monocytes to actively release HMGB1 J Leukoc Biol 81 2007 741 747 [23] A. Tsung J.R. Klune X. Zhang HMGB1 release induced by liver ischemia involves toll-like receptor 4-depedent reactive oxygen species production and calcium-mediated signaling J Exp Med 204 2007 2913 2923