The association of novel inflammatory marker GlycA and incident atrial fibrillation in the Multi-Ethnic Study of Atherosclerosis (MESA)

Background Emerging evidence has implicated that inflammation contributes to the pathogenesis of atrial fibrillation (AF). GlycA is a novel marker of systemic inflammation with low intra-individual variability and high analytic precision. GlycA has been associated with incident cardiovascular disease (CVD) independent of other inflammatory markers. However, whether GlycA is associated with AF, specifically, has yet to be established. We examined the association between GlycA and AF in a multi-ethnic cohort. Methods We studied 6,602 MESA participants aged 45–85, with no clinical CVD at baseline, with data on GlycA and incident AF. We used multivariable-adjusted Cox models to evaluate the association between GlycA and incident AF. We also examined other inflammatory markers [high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL6) and fibrinogen] and incident AF for comparison. Results The mean (SD) age was 62 (10) years, 53% women. The mean plasma GlycA was 381 (62) μmol/L. Over median follow-up of 12.9 years, 869 participants experienced AF. There was no statistically significant association between GlycA and incident AF after adjusting for sociodemographics, CVD risk factors, and other inflammatory markers [Hazard Ratio (95% CI) per 1 SD increment in GlycA: 0.97 (0.88–1.06)]. Neither hsCRP nor fibrinogen was associated with incident AF in same model. In contrast, IL-6 was independently associated with incident AF [HR 1.12 per 1 SD increment (1.05–1.19)]. Conclusions Although GlycA has been associated with other CVD types, we found that GlycA was not associated with AF. More research will be required to understand why IL-6 was associated with AF but not GlycA. Clinical trial registration MESA is not a clinical trial. However, the cohort is registered at: URL: https://clinicaltrials.gov/ct2/show/NCT00005487 Unique identifier: NCT00005487.

a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 the datasets and have to keep them stored securely in a Cloud using encrypted software for protection. However, other researchers may obtain the minimal dataset required to replicate our study's findings by submitting a proposal through the NIH BioLINCC (https://biolincc.nhlbi.nih.gov/studies/ mesa/). Additionally, one can apply to the MESA Coordinating Center to become a new investigator after signing a Data Use Agreement. Please direct inquires about MESA data access to Craig Johnson (wcraigj@uw.edu) at the MESA Coordinating Center or at https://www.mesa-nhlbi.org/. Our findings should be easily reproducible through the methods described in this paper.
Funding: The MESA study was supported by contracts HHSN268201500003I, N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, and R01 HL127659 from the National Heart, Lung, and Blood Institute (NHLBI), and by grants UL1-TR-000040, UL1-TR-001079, and UL1-TR-001420 from the National Center for Advancing Translational Sciences. EDM and DZ are additionally funded by the Blumenthal Scholars Award in Preventive Cardiology at Johns Hopkins University. SJ was supported by the Dean's Research Fund at Johns Hopkins University School of Medicine. The aforementioned NHBLI funding agency provided financial support of the study for participant recruitment and data collection, as well as support in the form of salaries for authors OO, SRH, MS, and EDM (paid to their respective institution). The aforementioned Johns Hopkins funding source provided support in form of salaries for authors EDM, DZ, and SJ. The commercial affiliation Laboratory Corporation of America Holdings (LabCorp) provided support in the form salary for JDO. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the 'author contributions' section. associated with AF, specifically, has not been well established. This is important to evaluate because GlycA may be a prognostic marker for AF or a target of therapy in the future.
To address this knowledge gap, we sought to determine the association of baseline GlycA and AF in a large multi-ethnic cohort free of overt clinical CVD at baseline. We hypothesized that higher plasma levels of baseline GlycA are associated with increased incidence of AF, independent of other traditional risk factors and other common inflammatory markers.

Transparency and Openness Policy (TOP)
The Multi-Ethnic Study of Atherosclerosis (MESA) participates in the NIH BioLincc Open program, and requests for access to MESA data can be submitted to: https://biolincc.nhlbi.nih. gov/studies/mesa/. Additionally, other researchers can apply to the MESA Coordinating Center to become a new investigator after signing a Data Use Agreement (DUA); more details are available at https://www.mesa-nhlbi.org/. Upon receipt of a DUA with the MESA Coordinating Center, access to the de-identified MESA databases can be made available to other researchers with approved proposals. Our findings should be easily reproducible through the methods described in this paper.

Study sample
MESA is an ongoing prospective cohort study that recruited ethnically diverse individuals free of clinically recognized CVD, including AF, and apparently healthy at baseline from 6 field centers in the United States to assess the characteristics of subclinical CVD, and risk factors for its progression. According to the MESA study protocol, individuals with clinically recognized CVD were excluded from the study. This refers to people who had physician-diagnosed heart attack, angina, stroke, transient ischemic attack, heart failure, or atrial fibrillation, those taking nitroglycerin and those who underwent procedures related to CVD (coronary artery bypass grafting, angioplasty, valve replacement, pacemaker or defibrillator implantation, any surgery on the heart or arteries).
The study initially enrolled a total of 6,814 individuals aged 45 to 84 years, 38% White, 28% African, 22% Hispanic, and 12% Asian between 2000 and 2002. Since the initial visit, 5 subsequent visits have taken place. At each visit, participant demographics, medical history, and physical examination results were collected. Further details about the study design have been reported [24].
In our analysis, individuals missing either GlycA measurements at baseline or incident AF follow-up data were excluded, leaving a total of 6,602 participants for inclusion (Fig 1).

Ethical approval statement
The MESA study was approved by the institutional review boards (IRB) at each participating center and each study participant provided informed consent. At Johns Hopkins, our study was approved by the Johns Hopkins School of Medicine IRB applicant number NA_00030361.

Exposure assessment
GlycA was analyzed from EDTA plasma samples, which were obtained from MESA baseline visit (2000)(2001)(2002) and stored at -70˚C until measured using NMR LipoProfile 1 as previously described [21,23,25]. The NMR assay detects the level of glycan residues of acute phase proteins and quantifies GlycA levels, which are expected to be elevated during inflammation [17]. The intra-assay and inter-assay coefficients of variance (CV) for NMR LipoProfile 1 were 1.9% and 2.6%, respectively. GlycA levels haven been shown to be similar when measured after short-term vs. long-term storage, in plasma vs. serum samples, and fasting vs. non-fasting state [17].

Covariates
Using data obtained from the baseline MESA visit, we considered demographics (age, sex, race/ethnicity, and MESA site), behavioral factors (smoking status, pack-years of smoking, and physical activity), socioeconomic factors (education, health insurance), adiposity [Body Mass Index (BMI)], traditional CVD risk factors [systolic blood pressure (SBP), use of antihypertensive medication, total cholesterol, HDL-cholesterol, use of lipid-lowering medication, diabetes and estimated glomerular filtration rate (eGFR)], and other inflammatory markers (hsCRP, IL-6, fibrinogen) for covariate adjustment. Height, weight, and blood pressure were measured following the protocol. BMI was calculated as weight divided by squared height (kg/ m 2 ). Physical activity was estimated in metabolic equivalent minutes per week using a 28-item Typical Week Physical Activity Survey [26]. Baseline blood pressure was measured 3 times in the seated position with the Dinamap automated blood pressure device, and the average of the last 2 measurements were used. Glomerular filtration rate was estimated using the formula from the Chronic Kidney Disease Epidemiology Collaboration [27]. Diabetes was defined as fasting blood glucose level �126 mg/dl, self-reported diagnosis of diabetes, or use of diabetes medication. Inflammatory biomarkers, including hsCRP, IL-6, and fibrinogen were measured from stored serum samples obtained at the baseline examination [18,28].

Outcomes assessment
Prevalent AF identified by study visit electrocardiogram (ECG) or self-report at baseline was an exclusion criterion for enrollment in the study. After enrollment, trained staff called study participants or their proxy every 9 to 12 months to identify all new hospitalizations and obtained discharge diagnostic codes. Participants were followed until July 15, 2015. Incident AF was ascertained by 1) study ECGs at a follow-up visit consistent with AF; 2) hospital discharge diagnoses [International Classification of Diseases, Ninth Revision, Clinical Modification code 427.31 (AF) or 427.43 (atrial flutter)] for AF; or 3) Medicare inpatient and outpatient claims data for individuals enrolled in fee-for-service Medicare. Previous work has shown the validity (up to 89%) of discharge diagnoses for identifying AF [29].

Statistical analysis
Baseline GlycA, hsCRP, IL-6, and fibrinogen levels were modeled in quartiles and also continuously per one standard deviation (SD) increment. Baseline characteristics of study population were presented by incident AF status. Kaplan-Meier curves were plotted of AF-free survival by GlycA quartiles. Multivariable-adjusted Cox proportional hazard regression models were used to estimate hazard ratios (HRs) and their 95% confidence intervals (CIs) for the association of incident AF by GlycA levels.
We evaluated 4 progressively adjusted models. In model 1, we adjusted for demographics and study site (age, sex, race/ethnicity, MESA site). Model 2 adjusted for model 1 covariates with the addition of socioeconomic, behavioral, and adiposity measures (education, health insurance, smoking status, pack-years of smoking, physical activity, and BMI). Model 3 additionally adjusted for CVD risk factors that may be intermediate variables between inflammation/adiposity and AF risk (SBP, use of antihypertensive medication, total cholesterol, HDLcholesterol, use of lipid-lowering medication, diabetes and eGFR). In model 4, we further adjusted for other commonly studied inflammatory markers, specifically log-transformed hsCRP, IL-6, and fibrinogen. In the same fully adjusted model (model 4), we also evaluated the association of the other inflammatory markers with incident AF, to compare with GlycA. In a supplemental analysis, we further adjusted for incident heart failure and incident coronary heart disease (CHD) in addition to all covariates adjusted for in model 3.
In addition, we used restricted cubic spline adjusted for the variables in Model 4 with knots placed at the 5th, 35th, 66.5th, 95 th percentiles to characterize the nonlinear association between baseline GlycA (continuous) levels with incident AF. We also examined multiplicative interactions of baseline GlycA (and the other inflammatory markers) with AF by age, sex, and race/ethnicity categories. The analyses were performed using STATA version 15.0 (StataCorp LP, College Station, TX). P values were two-sided, with significance level set at 0.05.

Baseline characteristics
The baseline characteristics of the 6,602 participants included in the analyses are shown in Table 1 by incident AF status and S1 Table by baseline GlycA level quartiles. Among the sample, the mean (SD) for age was 62 (10) years with 53% being women, 38% White, 27% Black, 22% Hispanic, and 12% Chinese. The mean (SD) for baseline plasma GlycA level was 381 (62) μmol/L. Those with higher GlycA levels tended to be women, have a lower educational background, and have a higher BMI, SBP, and prevalence of diabetes mellitus. In addition, those with higher GlycA levels tended to have a higher median level of inflammatory biomarkers, such as hsCRP, IL-6, and fibrinogen (S1 Table).
Over a median (IQR) follow-up time of 12.9 (10.0-13.6) years, a total of 869 participants (13%) experienced AF. Those who developed AF tended to be men, White, and have a higher age, median pack-years of smoking, SBP, and a greater prevalence of diabetes mellitus, and were more likely to take medications for hypertension and hyperlipidemia. They had lower average physical activity levels and eGFR, although BMI was similar to those who did not develop AF. Participants with AF had similar levels of baseline GlycA, hsCRP, and fibrinogen; however, baseline IL-6 levels were higher among those who developed incident AF ( Table 1).

Associations of GlycA with incident AF
The incidence rates and HRs for the association of baseline GlycA with incident AF are shown in Table 2. In unadjusted analysis, there was no significant difference in survival free of AF by baseline GlycA quartiles (Fig 2, log-rank test: P = 0.64). There remained no significant association of GlycA with incident AF in multivariable adjusted models. After adjusting for demographics, such as age, sex, race/ethnicity, and MESA site (model 1), compared to the lowest  association of baseline GlycA and incident AF. In the restricted cubic spline model, the association of baseline GlycA with risk of AF appeared U-shaped; however, confidence intervals were wide and associations not statistically significant (Fig 3).
Given that GlycA has already been demonstrated to be associated with incident heart failure and CHD, we performed a supplemental analysis to determine whether GlycA was still associated with AF after adjusting for interim heart failure event or CHD in addition to all covariates adjusted for in model 3 (S2 Table). In this supplemental analysis, the adjusted HR (95% CI) for AF per 1 SD higher GlycA was 0.96 (0.89-1.04), still showing no statistically significant association between GlycA and AF.

Associations of hsCRP, IL-6, and fibrinogen with incident AF
Incidence rates and HRs for the association of other inflammatory biomarkers at baseline, such as hsCRP, IL-6, and fibrinogen with incident AF were also evaluated among the 6,446 individuals with complete biomarker assessment (Table 3). There was no significant association of hsCRP with incident AF with adjusted HRs (95% CI) of 1.00 (0.82-1.  with AF. Compared to the lowest IL-6 quartile, the adjusted HRs (95% CI) for AF were 1.05 (0.84-1.32), 1.33 (1.06-1.67) and 1.42 (1.10-1.82) for the 2 nd , 3 rd and 4 th quartiles respectively, and was 1.12 (1.05-1.19) per 1 SD increment in IL-6. Interactions by age, race/ethnicity and sex were not significant for hsCRP, IL-6 and fibrinogen.

Discussion
Contrary to our hypothesis, in this analysis from a diverse community cohort free of CVD at baseline, we found that higher concentrations of a novel composite inflammatory biomarker GlycA at baseline was not associated with greater incidence of AF over a 12.9-year median follow-up. Given that GlycA was a composite marker reflective of multiple acute phase proteins, we had hypothesized that it would be a superior marker of predicting AF risk compared to the other inflammatory markers. Rather, we only found IL-6 to be independently associated with incident AF.
We investigated this analysis because previous research has found GlycA to be associated with other diseases with an inflammatory etiology, including auto-immune diseases [30][31][32] and other types of CVD. In a prior MESA study, higher levels of GlycA were associated with having a greater prevalence of an unfavorable cardiovascular health profile, and with poorer status of many of the individual components of the American Heart Association Life Simple 7 score (i.e. BMI, physical activity, smoking, blood pressure, glucose, cholesterol)-health metrics which are also risk factors for AF [18]. Higher GlycA levels have been linked with subclinical Table 3. Incidence rates (95% CI) and hazard ratios (95% CI) for the association of CRP, IL-6 and fibrinogen with incident atrial fibrillation: The multi-ethnic study of atherosclerosis (2000-2015). atherosclerosis [30,33,34], incident CVD [19][20][21][22], and with heart failure with preserved ejection fraction [23]. In a prior MESA study comparing the predictive value of GlycA and other inflammatory biomarkers, GlycA had a significantly predictive value comparable to hsCRP, IL-6, and D-dimer, if not superior, for total death, CVD, chronic inflammatory-related severe hospitalization and death, and total cancer [21]. Thus, we had hypothesized that GlycA might be a superior marker for AF too, although our findings came to a different conclusion. Of note, prior work in MESA has found that GlycA is only modestly to moderately correlated with other markers of inflammation [with Pearson correlation coefficient for GlycA with ddimer (0.09), IL-6 (0.29), hsCRP (0.47), and fibrinogen (0.49) [18], and thus we had anticipated associations with AF independent of other inflammatory markers.

CRP in mg/
There is extensive evidence that inflammation plays a key role in the development of AF. The association of some inflammatory biomarkers with AF had been studied, but the relationship between GlycA, specifically, and AF had not been explored before our report. Consistent with our results, in a recent Chronic Renal Insufficiency Cohort (CRIC) study that assessed the association between common inflammatory biomarkers (hsCRP, fibrinogen, TNFα, and IL-6) and AF, only plasma IL-6 level was significantly associated with both the presence of AF at baseline and new-onset of AF in chronic kidney disease patients [35]. A prior MESA study that evaluated the prognostic value of IL-6 for various CVD outcomes found the association of IL-6 with AF appeared greater among statin users compared to non-users [36]. In contrast to our findings, in that analysis there was no longer a statistically significant association of IL-6 with AF in multivariable-adjusted models, but differences between that study and ours may be their stratification by statin use at baseline which reduced statistical power, and we additionally adjusted for multiple inflammatory markers including GlycA which was not included in their models [36].
Other inflammatory markers have been linked to AF in prior work. One study examined the relationship between collagen biomarkers and AF and demonstrated that high levels of plasma collagen biomarkers were also associated with excess risk for AF [37]. Alegret et al showed that CRP and C-C Motif Chemokine Ligand 2 (CCL2) concentrations were significantly increased in AF patient compared to healthy subjects [38]. However, when patients with paroxysmal AF and permanent AF were separately analyzed, while increase in CCL2 was observed in both subgroups, CRP was only elevated in those with the permanent condition. Further analysis of the genetic variants of CRP and CCL2 did not support strong association of the inflammatory biomarkers and AF, leading to the conclusion that the duration of the episode is important to consider in assessing the role of inflammation, and that elevation of CCL2 is the consequence, not the cause of AF.
Together, these collective studies suggest the relationship between AF and the process of inflammation is multifactorial and complex. High levels of inflammatory biomarkers and inflammatory cells such as neutrophils and lymphocytes have been observed in patients with AF. When local or systemic inflammation occurs, it induces atrial electrical and structural remodeling, which triggers AF. In turn, AF promotes inflammation by mechanisms yet to be understood, and perpetuates the arrhythmia [2]. Markers reflective of acute inflammation vs. chronic inflammation may have differing associations with AF. While inflammation has been implicated in the pathophysiology of AF, and several inflammatory biomarkers have been proposed to be associated with AF, the role of inflammation in guiding AF management has not been well established.

Strengths and limitations
Our study must be interpreted in the context of several limitations. First, due to our ascertainment methods, paroxysmal cases of AF that were not present at study visits or did not result in hospitalization may have been missed. Second, incident AF cases were ascertained from hospitalization discharge codes, which could have been misclassified. However, these codes have been demonstrated to have adequate positive predictive value for the ascertainment of AF events [39]. Third, while we included several covariates that likely are to influence the development of AF in the statistical models, residual confounding remains a possibility. Fourth, GlycA levels were measured only once at baseline, and thus we were unable to assess change in GlycA levels over time. Previous work has shown that a single baseline measure could accurately capture the short-term inflammatory status over four to 6 months [40]. However, we do not know if the same accuracy holds for longer periods of time.
Despite these limitations, our study had a number of strengths. We used data from a large multiethnic, multiracial cohort of patients without clinical CVD at baseline who were followed longitudinally with a median follow up of 12.9 years to determine development of AF. We were also able to examine multiple biomarkers for inflammation other than our primary biomarker of interest, GlycA, for comparison in the same population. To our knowledge, our study was the first to examine the association of GlycA with AF.

Conclusions
In summary, we found that elevated GlycA levels at baseline were not associated with higher risk of incident AF, but IL-6 levels were. This suggests that GlycA is not a prediction marker for AF. Further research is required to understand why the inflammatory marker IL-6, but not GlycA, was associated with AF. Several inflammatory biomarkers have been proposed to be associated with AF, but due to the complex pathophysiology of AF each biomarker's predictive value in guiding AF management has not been well established. Nevertheless, it is undeniable that inflammation contributes to the pathological process of AF. A better understanding of inflammatory markers related to AF improves understanding of the pathophysiology of the disease as well as its risk prediction.
Supporting information S1  Table. Incidence rates (95% CI) and hazard ratios (95% CI) for the association of GlycA with incident atrial fibrillation, adjusted for incident heart failure and coronary heart disease.