Blood-based 8-hydroxy-2 0 -deoxyguanosine level: A potential diagnostic biomarker for atrial ﬁ brillation

BACKGROUND Recent research ﬁ ndings have revealed a key role of oxidative DNA damage in the pathogenesis of atrial ﬁ brillation (AF). Therefore, the circulating oxidative DNA damage marker 8-hydroxy-2 0 -deoxyguanosine (8-OHdG) may represent a biomarker for staging AF and identifying patients at risk for AF recurrence and postoperative atrial ﬁ brillation (POAF) after treatment. OBJECTIVE The purpose of this study was to investigate whether serum levels of 8-OHdG correlate with the stage of AF, recurrence after AF treatment, and onset of POAF after cardiac surgery. METHODS In this prospective observational study, 8-OHdG levels were detected by enzyme-linked immunosorbent assay in human serum samples. Blood samples were collected from control patients without AF history; patients with paroxysmal AF and persistent AF undergoing electrical cardioversion or pulmonary vein isolation (PVI); and patients with sinus rhythm (SR) undergoing cardiac surgery. AF recurrence was determined during 12-month follow-up. Univariate and multivariate analyses were used to identify changes in 8-OHdG levels between the groups. RESULTS Compared to the control group, 8-OHdG levels in the patient groups gradually and signi ﬁ cantly increased during arrhythmia progression. 8-OHdG levels in AF patients showing AF recurrence after PVI treatment were signi ﬁ cantly increased compared to patients without AF recurrence. Moreover, in SR patients undergoing cardiac surgery, 8-OHdG levels were signi ﬁ cantly elevated in those showing POAF compared to patients without POAF. CONCLUSION 8-OHdG level may represent a potential diagnostic biomarker for AF staging as well as for predicting AF recurrence and POAF after treatment.


Introduction
Atrial fibrillation (AF) is the most common progressive tachyarrhythmia and is associated with serious complications such as heart failure and stroke. 1 Due to the aging population, the prevalence of AF is steadily rising, especially in the population between 75 and 85 years of age. 2 AF increases morbidity and mortality rates, with a 5-fold increased risk of stroke and a 3-fold increased risk of congestive heart failure. 2 Therefore, AF contributes to medical and economic problems worldwide. 3,4 In addition, 30%-50% of patients who undergo cardiac surgery develop postoperative atrial fibrillation (POAF). 5,6 POAF frequently occurs within 5 days after surgery, with a peak incidence on postoperative day 2. 5,7 Patients who develop POAF may require a longer period of hospitalization because POAF is associated with a higher risk of other postoperative complications, such as stroke and perioperative myocardial infarction. 5,8 Early recognition and staging of AF are essential to initiate treatment to prevent disease progression. AF progression is accompanied by a congruent increase in therapy failure and in the end stage cannot, even with extensive therapy, be treated. [9][10][11] At present, AF can only be diagnosed with a surface electrocardiogram when the patient has already experienced AF. However, no effective diagnostic tool is available for staging AF. Therefore, diagnostic biomarkers that can stage and possibly predict AF are clinical highly relevant. 10 Emerging evidence indicates that AF is intimately linked with DNA damage in experimental AF models and in patients with persistent AF. A recent study using experimental AF models revealed that electrical and contractile impairment of atrial cardiomyocytes is precipitated by nicotinamide adenine dinucleotide (NAD 1 ) depletion via excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. 12 PARP1mediated synthesis of ADP ribose chains, in turn, depletes NAD 1 levels, thereby inducing further oxidative DNA damage and electrical and contractile dysfunction, which drives AF progression. 12,13 Consistent with these findings, cardiomyocytes of patients with persistent AF show significant oxidative DNA damage in atrial tissue, characterized by an elevated level of phosphorylated histone 2A family member X (H2AX), forming g-H2AX, which is an early response to the induction of DNA double-strand breaks. 12,14,15 Based on these findings, we hypothesize that oxidative DNA damage represents a potential biomarker for staging AF and predicting AF recurrence. Interestingly, 8-hydroxy-2ʹ-deoxyguanosine (8-OHdG) is an important byproduct of oxidative DNA damage, which is formed during the repair of damaged DNA by exonucleases. 16 As 8-OHdG is circulating in the blood after oxidative damage in DNA, it may represent a biomarker for AF. 17 In this study, we correlated serum 8-OHdG levels with AF stage and recurrence after AF therapy, and tested whether 8-OHdG predicts POAF in patients with sinus rhythm (SR) undergoing cardiac surgery.

Patient characteristics
In this prospective observational study, 257 serum samples were collected from patients 1 day before cardiac surgery, pulmonary vein isolation (PVI), or electrical cardioversion (ECV) and from control patients at the cardiology department at the Erasmus Medical Center (Rotterdam, The Netherlands). To study 8-OHdG levels in various AF stages, the following groups were included: control group (control; N 5 50), paroxysmal AF group (PAF; N 5 74), and persistent AF group (PeAF; N 5 89). To study 8-OHdG levels in POAF, a group of patients in SR undergoing cardiac surgery (N 5 44) were included. Detailed clinical characteristics of the control and AF patients are given in the Supplemental Methods. All AF patients were treated with anticoagulants and signed consent forms before inclusion in the study. This substudy is part of the HALT & REVERSE (Hsf1 Activators Lower Cardiomyocyte damage: Towards a novel approach to REVERSE atrial fibrillation) trial (Rotterdam Medical Ethical Committee MEC-2014-393) and is approved by the institutional medical ethical committee. The study was performed according to the principles of the Declaration of Helsinki and in accordance with the Medical Research Committee Involving Human Subjects Act.

DNA damage marker 8-OHdG measurement
Blood samples were collected for measurement of the DNA damage marker 8-OHdG. Details are available in the Supplemental Methods.

Statistical analysis
Quantitative data are given as mean 6 SD. Statistical analyses were performed using SPSS Statistics Version 26.0 for Windows (SPSS, Chicago, IL) and GraphPad Prism Version 8.0 (GraphPad Software, San Diego, CA). If data were not normally distributed, then log10 transformation was performed before statistical analysis. One-way analysis of variance (ANOVA) and post hoc Tukey tests were used to assess 8-OHdG levels in each group. Two-way ANOVA and post hoc Tukey tests were used to assess sex differences. Differences in clinical characteristics between control, PAF, and PeAF were tested with 1-way ANOVA, Kruskal-Wallis test, and c 2 test. Differences in clinical characteristics between patients with and those without AF recurrence were tested with independent-samples Student t test, Mann-Whitney test, and c 2 test. Levels of 8-OHdG in patients with or those without AF recurrence in the AF group and levels of 8-OHdG in patients with or those without POAF in the SR group were compared using the nonparametric Mann-Whitney test. Linear regression was used for age, body mass index (BMI), hypertension, diabetes, and antiarrhythmic drug class I (AAD-I) correction. A receiver operating characteristic (ROC) curve was used to analyze the diagnostic power of 8-OHdG. All P values were 2-sided, and P ,.05 was considered significant.

Clinical characteristics
To study the correlation between 8-OHdG and AF stage, 213 patients were included in the study: a control group of patients (N 5 50 [23%]) without AF and a study group of 163 AF patients with either PAF (N 5 74 [35%]) or PeAF (N 5 89 [42%]). Table 1 lists the characteristics of the study population. Patients with AF were older (P ,.05), presented with higher BMI (P ,.05), more often had hypertension and diabetes (P ,.05), had a larger left atrial volume (P ,.05), and presented with more impaired left ventricular function (P ,.05) compared to control patients without AF.

Serum levels of 8-OHdG stage AF
To test whether oxidative DNA damage is associated with AF stage, we measured the level of 8-OHdG in the serum samples of the patient population (Table 1). Compared to the control patients, serum 8-OHdG levels were gradually and significantly increased in PAF and PeAF patients (control vs PAF: 6.76 6 2.77 ng/mL vs 8.33 6 4.11 ng/mL; P 5 .018; PAF vs PeAF: 8.33 6 4.11 ng/mL vs 10.52 6 4.22 ng/mL; P 5 .001) even after correction for age, BMI, hypertension, and diabetes with linear regression ( Figure 1A). No differences in 8-OHdG levels were observed between males and females ( Figure 1B). The findings indicate that the oxidative DNA damage marker 8-OHdG gradually increases in more advanced stages of AF and that this effect is sex independent.

Diagnostic value of 8-OHdG as a blood-based biomarker for AF
To determine whether the serum level of 8-OHdG has power to discriminate AF patients from controls, an ROC curve was constructed. Figure 2 shows an area under the curve (AUC) of 71%, which corresponds to sensitivity of 80% and specificity of 44%. These findings indicate that 8-OHdG levels have value in discriminating between AF patients and controls and therefore may represent an independent and valuable diagnostic biomarker for AF.

Relation between 8-OHdG levels and AF recurrence
To investigate a relation between serum 8-OHdG levels and AF recurrence after PVI or ECV treatment, we compared 8-OHdG levels in the AF population between patients with and those without AF recurrence after treatment. Clinical parameters are listed in Table 2. In the PVI group, patients with AF recurrence after PVI treatment presented more often with hypertension (P ,.05) than did patients without AF recurrence. However, patients without AF recurrence after PVI treatment more often received treatment with AAD-I (P ,.05) compared to patients with AF recurrence. In the ECV group, patients with AF recurrence after ECV treatment presented more often with hypertension (P ,.05) than did patients without AF recurrence ( Table 2). In addition, 54% of PVI patients and 59% of ECV patients experienced AF recurrence within 12 months. Levels of 8-OHdG in patients with AF recurrence after PVI treatment are significantly higher (no recurrence vs recurrence: 7.35 6 3.23 ng/mL vs 9.55 6 4.14 ng/mL; P 5 .018) compared to patients without AF recurrence, even after correction for hypertension and AAD-I treatment with linear regression ( Figure 3A). No differences in serum levels of 8-OHdG was found in AF patients with AF recurrence after ECV treatment compared to those without AF recurrence (no recurrence vs recurrence: 9.76 6 3.09 ng/mL vs 10.99 6 4.72 ng/mL; P 5 .214) ( Figure 3B).

Relation between 8-OHdG levels and POAF
Oxidative stress is an important contributor to POAF. 18,19 Therefore, we evaluated 8-OHdG levels in SR patients undergoing cardiac surgery and correlated the levels with development of POAF. Characteristics of the study population are listed in Table 3. Clinical parameters, including sex, age, and left atrial dilation were comparable between SR patients with and those without POAF after cardiac surgery. Compared to SR patients without POAF, 8-OHdG levels in patients with POAF were significantly increased (9.76 6 5.42 ng/mL vs 10.82 6 5.58 ng/mL; P ,.001) (Figure 4). This finding indicates that preoperative 8-OHdG levels are Values are given as n (%) or mean 6 SD unless otherwise indicated. correlated with POAF and therefore may represent a potential predictor of POAF in SR patients undergoing cardiac surgery.

Discussion
In this study, we observed that levels of the oxidative DNA damage marker 8-OHdG before PVI or ECV treatment were significantly increased during more persistent stages of AF. Also, 8-OHdG levels in patients with an AF recurrence after PVI treatment were significantly increased compared to patients without AF recurrence within 12 months of follow-up. Importantly, the level of 8-OHdG is independent of patient characteristics in all groups studied. Moreover, SR patients undergoing cardiac surgery had elevated levels of 8-OHdG in case of POAF onset compared to patients without POAF. Thus, our findings indicate that 8-OHdG levels may have diagnostic value in staging AF and predicting AF recurrence after PVI and POAF onset following cardiac surgery.

Serum 8-OHdG level as potential biomarker for AF
Although AF is diagnosed with an electrocardiogram, the stage of AF cannot be determined. Previous biomarker studies reported associations between cardiac troponin T, troponin I, natriuretic peptide, transforming growth factorbeta, and tissue inhibitor of metalloproteinase-1 levels with AF. [20][21][22][23][24] However, whether these biomarkers could stage AF and predict AF recurrence after PVI treatment is unknown. 25 Our previous study identified a key role of oxidative DNA damage in driving experimental and clinical AF. 12 Based on these mechanistic findings, we investigated oxidative DNA damage levels in serum samples of AF patients. In line with the mechanistic DNA damage study, the current study reports that the marker for oxidative DNA damage, 8-OHdG, can discriminate 3 relevant AF patient populations from controls. These 3 discriminated AF populations include patients in the various AF stages from controls without AF; patients with an AF recurrence after PVI from patients without recurrence within 12 months of follow-up; and patients who develop POAF from patients without POAF.  To date, only 1 previous study has reported on the role of 8-OHdG as a biomarker for AF. In that study, 8-OHdG levels were found to be increased in urine samples of AF patients compared to controls in SR, and the levels were reduced after ECV or ablative therapy. 26 No data on prediction of AF recurrence and POAF have been reported to date. Interestingly, in the current study, ROC curve values indicate that the level of 8-OHdG in serum has Values are given as n, n (%), or mean 6 SD unless otherwise indicated. AF 5 atrial fibrillation; other abbreviations as in Table 1. sufficient power to discriminate AF patients from controls. Therefore, serum 8-OHdG levels may have diagnostic value as a biomarker for identifying AF stage and recurrence in patients.

Serum 8-OHdG levels predict POAF
The etiology of POAF is considered to be multifactorial and involves a complex interaction of "triggering" stimuli and "sustaining" processes acting on a myocardial substrate that may be predisposed to developing AF. 27 Recently, inflammation and oxidative damage have been identified as key contributors to POAF. 18,27 Surgical trauma, ischemia during both cardiopulmonary bypass and cardioplegic arrest, and reperfusion contribute to induction of oxidative damage and the production of proinflammatory molecules including interleukin-6, tumor necrosis factor-a, and C-reactive protein. [27][28][29] Moreover, cardiopulmonary bypass and cardioplegic arrest-related oxidative stress may trigger cellular changes in atrial tissue and lead to disruption of electrical activity. 18,30 The main atrial remodeling associated with the pathogenesis of POAF is production of reactive oxygen species, primarily via enhanced NADPH oxidase activity due to mitochondrial dysfunction in atrial cardiomyocytes. 31,32 Interestingly, in the current study, preoperative 8-OHdG levels in SR patients who developed POAF after cardiac surgery were significantly increased compared to patients without POAF. This suggests that oxidative DNA damage may be associated with onset of POAF, so these levels may predict the chance of development of POAF in SR patients after cardiac surgery. Future research is warranted to confirm this finding and elucidate the exact mechanism of how the oxidative DNA damage pathway may contribute to POAF onset.  Values are n (%) or mean 6 SD unless otherwise indicated. AVD 5 aortic valve disease; CABG 5 coronary artery bypass graft; CAD 5 coronary artery disease; MVD 5 mitral valve disease; POAF 5 postoperative atrial fibrillation; SR 5 sinus rhythm; other abbreviation as in Table 1. AF-related markers. Additional markers may include phosphorylated H2AX, NAD 1 , and NADH levels.

Conclusion
The level of 8-OHdG is closely linked with the stage of AF, as the level gradually increases during more advanced stages of AF. 8-OHdG levels may predict AF recurrence after ablative therapy and POAF onset after surgical treatment of cardiovascular diseases. The level of 8-OHdG may represent a potential diagnostic biomarker for staging AF and predicting AF recurrence after ablative therapy and POAF after cardiac surgery.

ERRATUM
In the article titled "Blood-based 8-hydroxy-2'-deoxyguanosine level: a potential diagnostic biomarker for atrial fibrillation" that was published in the February 2021 issue of Heart Rhythm (2021:18:271-277), Figure 1 was incorrect. The correct figure is given below. The publisher regrets this error.