CLINICAL ANALYSIS OF ASSOCIATION OF CYSTATIN C AND ATRIAL FIBRILLATION

Some studies have disclosed atrial fibrillation (AF) is associated with inflammation. Cystatin C is not only inflammatory markers but also an independent predictor of cardiovascular events. Aim. We sought to investigate the relationship between serum levels of cystatin C and the occurrence and development of AF. Material and methods. 235 paroxysmal and persistent AF (AF1 group) and 254 permanent AF (AF2 group) patients in AF group and 221 healthy people in control group were prospectively measured for cystatin C, other inflammatory markers, biochemical indicators, left atrial diameter (LAD), left ventricular diameter (LVD) and left ventricular ejection fraction (LVEF). Results. (1) Compared with control and AF1 groups, AF2 group had higher values of cystatin C, high sensitivity C reactive protein (hsCRP), LAD and LVD whereas lower values of LVEF (P<0.05). (2) After adjust for age, gender and body mass index (BMI), correlation analysis showed that serum level of cysta tin C was closely related to hsCRP, LAD, systolic blood pressure (SBP) and creatinine, the correlation coefficient were respectively 0,614, 0,520, 0,463 and 0,538 (all P<0,01), but negatively associated with LVEF (r= –0,356, P=0,012) in AF group. (3) Multivariate regression analysis showed the hsCRP, cystatin C, LAD and LVEF entered finally into the regression equation (cystatin C, OR: 3.25, 95%CI: 1.05–10.17, P=0,008). Conclusion. The serum levels of cystatin C has significant correlation with AF, which indicates cystatin C may play an important role in the process of AF development.


Introduction
Atrial fibrillation (AF) is the most common cardiac arrhythmia and increases in prevalence with aging [1][2][3].It has become one of the leading causes for hospitalized patients with AF because AF may induce stroke, heart failure and increase case fatality [1,2,4].Unfortunately, its fundamental pathological mechanisms are not fully clear.Recent evidence is accumulating that AF may be closely interrelated with inflammation and inflammatory biomarkers [2,[4][5][6].Some studies confirmed that diverse inflammatory factors participate in pathogenesis and development of AF [6][7][8].
Cysteine protease inhibitors-C (cystatin C) is a member of protease inhibitor superfamily.cystatin C is not only a relatively more sensitive indicator of evaluating renal function than creatinine, but also an independent and strong predictor of cardiovascular events [7,9].Recently studies find cystatin C is closely related to the inflammatory process or other inflammation factors [7,10].However, it remains challenged whether or not there is correlation between cystatin C and AF.In this study, the correlation between cystatin C and AF was investigated and its possible pathogenesis was preliminarily discussed and elucidated.

Patients
A total of 710 consecutively hospitalized patients with AF (assigned to group AF) were prospectively recruited between June 2008 and December 2010 from the Second Hospital of Shandong University and Qilu Hospital of Shandong University, which included 235 cases of paroxysmal and persistent AF (placed to group AF1), 121 males and 114 females with mean age of 67,42±12,29 years old.There were 254 cases of permanent AF (put to group AF2), 129 males and 125 females, averaged (68,15±11,52) years old.All cases of AF diagnosed were verified by medical history, physical examination, electrocardiogram or dynamic electrocardiogram.The control group had 221 cases of adults after health examination in the Second Hospital of Shandong University, selected from outpatients without diseases or with minor illnesses from cardiac or other departments following the same exclusion criteria.Of which, there were 113 male cases, 108 female cases with a mean age of 66,70±12,18 years old.Electrocardiogram showed sinus rhythm in control group.There were not any statistically significant differences (P>0,05) but comparability in comparison with age, sex and etiological composition among the three groups.
AF was defined and classified according to the management of atrial fibrillation of the European Society of Cardiology (ESC, 2010 edition) [11].Paroxysmal AF is self-terminating usually within 48 hours, and may continue for up to 7 days.Persistent AF is present when an AF episode either lasts longer than 7 days or requires termination by cardioversion, either with drugs or by direct current cardioversion.Permanent AF is said to exist when the presence of the arrhythmia is accepted by the patient (and physician).Patients with any of the following conditions were excluded from the study: infectious diseases, malignant tumors; hyperthyroidism; hypokalemia, hypomagnesemia, hypocalcemia and acidosis; pneumonia and pulmonary embolism; moderate and severe anemia; intracranial hemorrhage; liver and renal abnormal function and other organs dysfunction; immune system and endocrine metabolic diseases; pregnant women and breastfeeding women; on some medicines such as statins and angiotensin-converting enzyme-inhibitors and/or angiotensin II receptor blockers.This research was conducted in accordance with the ethical principles stated in the "Declaration of Helsinki".The study protocol and written informed consent were approved by the Ethics Committee of Clinical Research, the Secondary Hospital of Shandong University.

Methods
Peripheral vein blood were obtained from all participants early in the day after a 12 h fast, immediately transferred into a glass tube containing disodium ethylenediamine tetraacetic acid (EDTA), and centrifuged for 10 min at 3000 round/min, separated in aliquots and then stored at -80 °C.Cystatin C and hsCRP were respectively measured by means of a particle-enhanced turbidimetric immunoassay with commercial kits (Serum cystatin C, Beijing Leadman Biochemistry Co., Ltd.Beijing, China; hsCRP, Diagnostic System Laboratory Inc, Webster, TX, USA).Its normal reference value is 0-3mg/L.
Fasting blood glucose, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, blood urea nitrogen and serum creatinine were measured in automatic biochemical analyzer (Hitachi 7600, Tokyo, Japan) with enzymic method in all subjects.Blood routine was tested in a Sysmex XE-2100 hematology analyzer (Sysmex corporation, Kobe, Japan).Every participant received the test of a 12-lead MAC1200 electrocardiogram system (GE Healthcare, Milwaukee, WI, USA).Left atrium diameter (LAD), left ventricular diameter (LVD) and left vetricular ejection fraction (LVEF) were recorded using a Philips iE33 ultrasonocardiograph (Philips Medical Systems, Bothell, WA, USA).

Statistical treatment
Continuous variables were expressed as mean ± SD and categorical variables were presented as percentages.Continuous variables were compared using one-way ANOVA, and categorical variables were compared with chi-square test.The relationship between variables was evaluated by significance calculation of partial correlation analysis after adjusting classical risk factors (age, sex and body weight).The overall influence of selected risk factors on the AF was assessed using binary logistic regression.Predictors of AF were determined by the multivariate regression analysis.The association between variables and the occurrence of AF was represented by odds ratio (OR) and their accompanying 95% confidence interval (95% CI).SPSS 17.0 (SPSS Inc., Chicago, IL, USA) was used for all calculations.P < 0,05 was considered significant.

Comparison of baseline data between AF and control groups
Baseline characteristics are shown in Table1.Compared with control group, AF1 and AF2 groups did not have statistical significance in age, gender, triglycerides, total cholesterol and high-density lipoprotein cholesterol (P>0,05), but had higher values of BM, systolic blood pressure, fasting blood glucose and serum creatinine (P<0,05 or P<0,01).Values of blood urea nitrogen and low-density lipoprotein cholesterol were significantly higher whereas those of diastolic blood pressure were significantly lower in group AF2 than in control group.While there were not any significant differences in the values of blood pressure, blood urea nitrogen and lowdensity lipoprotein cholesterol between group AF1 and control group (P>0,05).Furthermore there were not any ORIGINAL STUDIES statistical differences in baseline datum between group AF1 and group AF2 (all P>0,05).

Comparison of inflammatory indicators between AF and control groups
As shown in Table 2, white blood cell counts showed no any significant difference (P>0,05) whereas there were significant difference in the values of cystatin C and hsCRP among these groups (P<0,05 or P<0,01).Compared with control group, AF1 and AF2 groups had higher values of cystatin C and hsCRP (P<0,05 or P<0,01).Furthermore group AF2 had higher values of cystatin C and hsCRP than those in group AF1 (P<0,05 or P<0,01).

Comparison of echocardiogram parameters between AF and control groups
As outlined in

Analysis for correlation between cystatin C and high risk factors of AF
As demonstrated in Table 4, after adjusting age, gender and body weight, cystatin C was closely related to hsCRP, left atrial diameter, systolic blood pressure and serum creatinine, and their correlation coefficient respectively were 0,614, 0,520, 0,463 and 0,528 (all P<0,01) whereas cystatin C was inversely related to left ventricular ejection fraction (r= -0,356, P=0,012).

Discussion
Cystatin C is a cysteine protease inhibitor with a molecular weight of 13kD, synthesized in all nucleated cells at a constant rate.It is present in an unglycosylated protein form and is ubiquitous in animal and plant tissue.It participate in proteolytic regulation between the interior and the exterior of the cell [7,12].Due to its free filtration in the glomerulus with nearly complete reabsorption and catabolism in the proximal tubule, and lack of tubular secretion, serum cystatin C concentrations are closely related to the glomerular filtration rate (GFR) reflecting renal function [7,9,12].So cystatin C is thought to be a specific, accurate and more sensitive marker than creatinine clearance rate.
In recent years, a large number of studies have confirmed that cystatin C is likely to be an independent risk factor of cardiovascular disease [7,12].The close relationship between cystatin C and cardiovascular disease is not only attributed to kidney function but is also thought to be mediated by inflammatory mechanism [7,13,14].The unique association of AF with renal dysfunction could be explained by the fact that AF and renal dysfunction share a number of risk factors [15].Although mechanical stress on atrium due to volume overload could be the mediating factor that leads to development of AF in patients with renal dysfunction, this does not explain the development of AF in earlier phases of renal dysfunction.In this situation, some researches make the beneficial attempt to these aspects [13,14].Both the ARIC Study [13] and the Heart and Soul Study [14] only demonstrated the association between AF and renal function estimated by urinary albuminto-creatinine ratio (ACR) and cystatin C-based glomerular filtration rate (eGFRcys) rather than cystatin C itself.The Malmö Diet and Cancer study [9] only provide evidence that natriuretic peptides and CRP instead of cystatin C improve prediction of incident heart failure and AF in the general population in addition to conventional risk factors.However, these studies [9,13,14] on cystatin C and AF are not discussed at all.One possible mechanism for a higher prevalence of AF in early stages of renal insufficiency could be explained by inflammation [15].In this study, as an indicator, only cystatin C among biochemical indicators (such as creatinine and blood urea nitrogen) refleting renal function in multivariate analysis had strong connection with AF.It is explained by the fact that cystatin C is more sensitive than other markers as a measure of renal function and is an inflammatory factor.Some researches also disclosed that cystatin C is a more reliable marker of renal function compared to creatinine or estimated GFR as it is less affected by age, gender, physical activity, diet and muscle mass, and ethnicity [9].
Many researchers have ascertained that cystatin C has a linear positive correlation with a variety of inflammatory cytokines such as hsCRP and reflects the severity of inflammatory activity, independent of renal function [12].Cystatin C and its fragments may also affect the phagocytic and chemotactic functions of granulocytes and participate in the inflammatory process [9,12].In atrial tissue of the patients with atrial fibrillation, inflammation results in inflammatory cell infiltration, oxidative stress and damage, this is followed by repair of the local tissue damage by fibrous tissue.As a result of this pathological process, atrial remodeling ensues [6][7][8].It is worth mentioning that Targoński et al. found that the serum concentration of hsCRP is closely, positively correlated with the diameter size of left atrium [16].The result of this study was consistent with Targoński's conclusion and showed that the serum concentration of cystatin C also coincided with left atrial diameter.This study confirmed that atrial fibrillation groups had higher values of cystatin C, hsCRP and left atrial diameter than those in the control group.Furthermore, persistent atrial fibrillation group had significantly higher values of cystatin C, hsCRP and left atrial diameter than those in paroxysmal AF and control groups.At the same time, correlation analysis showed that cystatin C is closely related to hsCRP and left atrial diameter of patients with atrial fibrillation.Therefore it follows that the inflammatory cytokines such as cystatin C and hsCRP should modulate process of inflammatory, participate in the hypertrophic degeneration of atrial muscle fiber, induce atrial structural abnormalities in patients with atrial fibrillation and thus lead to atrial electrical remodeling [6][7][8].Inflammation is closely associated to atrial fibrillation [6] and may be the important medium which links atrial fibrillation with known risk factors (such as high blood pressure and obesity, etc.) responsible for the development of atrial fibrillation [1,3,17].Even the atrial pathoanatomy in lone atrial fibrillation showed inflammatory infiltration, muscle cell necrosis and fibrosis [1,17].Modern research has confirmed that chronic inflammation has arrhythmogenic effect giving rise to the development of AF in susceptible populations.Inflammatory markers could be the result of atrial fibrillation rather than the cause of atrial fibrillation [18].Conen et al. found that a rise the augment of hsCRP increased the risk of AF by 31% in the elderly [18].In this study, monofactorial analysis showed that the serum levels of hsCRP and cystatin C in 2 atrial fibrillation groups were higher than those in control group, and they were closely related to each other.Multifactor analysis showed that both cystatin C and hsCRP, entered in the regression equation, had higher OR values (3.41 and 3.76, respectively).It was demonstrated that atrial fibrillation is closely associated with inflammation regardless of the duration of atrial fibrillation.However, this study showed no significant relationship between white blood cell count and risk of incident atrial fibrillation, which differs from the result of the Framingham Heart Study [19].
Cystatin C is not only an independent risk marker of predicting cardiovascular disease but also is an independent risk factor of MetS [7].As mentioned above, cystatin C was not only related to inflammation but also was associated with the risk factors of atrial fibrillation which are also the components of metabolic syndrome.The risk of developing AF in patients with metabolic syndrome increased by 88%, compared to those without metabolic syndrome. 3Atrial fibrillation and metabolic syndrome share common risk factors: obesity, hypertension, hyperglycemia and hyperlipidemia.The patients with higher level of cystatin C have higher metabolic state: higher body mass index, blood pressure, blood sugar and lipid levels [12].Researches have shown that cystatin C is closely related to the metabolic syndrome [3,7].Insulin resistance is not only the pathogenesis of metabolic syndrome but also may be the pathological process that connect cystatin C with metabolic syndrome [7,20].Presumably, from another perspective, atrial filbrallation and the metabolic syndrome may have a common pathological relationship mediated by inflammtory biomakers such as cystatin C.This study confirmed that body mass index, systolic blood pressure, fasting blood glucose and low-density lipoprotein cholesterol in AF groups, especially in permanent atrial fibrillation group (group AF2), were higher than those in the control group.Blood pressure is the most common risk factor of atrial fibrillation; moreover systolic blood pressure being the better predictor of atrial fibrillation than diastolic blood pressure [3].This study also revealed that systolic blood pressure closely correlated to cystatin C as showed in the univariate analyse.Linssen, et al. pointed out that AF facilitate the progression of heart failure in several ways.Due to rapid heart rates, an irregular ventricular rhythm, loss of atrioventricular synchrony, and an increase in mitral and tricuspid regurgitation, AF may further decrease cardiac output and aggravate heart failure [21].
As shown in this study, left ventricular ejection fraction was also independently aligned with AF.Some studies have validated that obesity is an independent risk factor for predicting atrial fibrillation [3].But this study showed that body mass index did not enter the regression equation in the multivariate analysis.

Study limitations
There were several limitations in this study.Our sample size, although small, was sufficient to diaplay differences between the control group and the group AF, however further studies with larger scale of cohorts are needed to confirmed these results.Additionally, some inflammatory indicators such as interleukin-6 and tumor necrosis factor -α were not applied in this study.Although these indicators maybe do not affect the conclusion of this study, which possibly made an impact on the estimate for action degree of hsCRP and cystatin C in this study.Furthermore, the relationship between cystatin C and atrial fibrillation was not verified by pathological and molecular biological methods.Finally, we did not progressively group the patients with paroxysmal and persistent atrial fibrillation into two parts according to AF duration.

Conclusion
In summary, as a new inflammatory factor, cystatin C is intimately associated with atrial fibrillation and may play an important role in the occurrence and development of atrial fibrillation.However, the specific relationship and precise mechanism between cystatin C and atrial fibrillation will need to be verified by a lot of further basic and clinical study.

Table 3 ,
Echocardiogram showed AF1 and AF2 groups had higher values of left atrial diameter and left ventricular diameter but lower values of left ventricular ejection fraction than those of control group (P<0,05 or P<0,01).Compared with group AF1, group AF2 had higher values of left atrial diameter and left ventricular diameter but lower values of left ventricular ejection fraction (P<0,05 or P<0,01).