Association between adiponectin‐to‐leptin ratio and heart rate variability in new‐onset paroxysmal atrial fibrillation: A retrospective cohort study

Abstract Background The adiponectin‐to‐leptin (A/L) ratio has been identified as a potential surrogate biomarker for metabolic disorders. However, it remains unknown whether the serum A/L ratio is associated with heart rate variability in paroxysmal atrial fibrillation (AF). Methods For this retrospective study, we included consecutive patients who underwent 24‐h long‐range electrocardiogram examination in our center for paroxysmal AF. The results of echocardiography, heart rate variability tests, and blood tests were also retrieved. Multivariate line regression analysis was performed to evaluate identify factors independently associated with heart rate variability. Results Among the 85 included patients with paroxysmal AF, the median A/L ratio was 1.71. Univariate analysis indicated that patients with a low A/L ratio (<1.71, n = 42) had a lower high‐frequency (HF) power and a higher hs‐CRP level, low‐frequency (LF) power, and LF/HF ratio than those with a high A/L ratio (≥1.71, n = 43). Multivariate linear regression analysis showed that the serum leptin concentration was independently and positively associated with LF (β = 0.175, p = .028), while the serum adiponectin concentration was independently and positively associated with HF (β = 0.321, p = .001). Moreover, the A/L ratio was independently and negatively associated with the LF/HF ratio (β = −0.276, p = .007). Conclusions The A/L ratio was independently and negatively associated with the LF/HF ratio in patients with new‐onset paroxysmal AF.

that obesity adversely affects cardiovascular hemodynamics, as well as cardiac structure and function, resulting in an increased prevalence of AF mediated by the altered secretion of a variety of adipokines such as leptin, adiponectin, and others (Lavie et al., 2017).
Adipokines play complex roles in the process of AF via their activities related to inflammation, autonomic function, and myocardial fibrosis (Kershaw & Flier, 2004;Lin et al., 2013;Prior Sarah et al., 2009;Zhou et al., 2020).
Adiponectin, one of the most abundant adipokines in human plasma, was shown to exert cardioprotective effects via its insulinsensitizing, anti-inflammatory, and antioxidant effects Ouchi et al., 2011;Prior Sarah et al., 2009). However, in patients with paroxysmal AF, a high circulating adiponectin level was confirmed to be independently associated with a poor clinical outcome after treatment by catheter ablation (Kim et al., 2018).
Additional experimental data indicate that interactions between adiponectin and the intrinsic cardiac autonomic nervous system may contribute to AF (Kershaw & Flier, 2004). Leptin is another important adipocytokine that regulates the physiological functions of the heart and has been linked to AF in the contexts of inflammation, myocardial remodeling, and sympathetic nervous system stimulation (Jenne & Tschopp, 1992;Jiang et al., 2020;Li & Liu, 2009;Lin et al., 2012).
Recent studies showed that the serum leptin/adiponectin ratio, which incorporates two major subgroups of adipokines, may offer prognostic value in many diseases based on findings that this ratio can reflect the presence of metabolic disturbances in obesity, the extent and severity of coronary artery disease, and idiopathic pulmonary fibrosis (Enomoto et al., 2019;Larsen et al., 2018;Rahmani et al., 2020). Vega and Grundy found that the adiponectin/leptin (A/L) ratio is a marker of adipose tissue dysfunction (Lena & Grundy, 2013), and Frühbeck et al. reported a negative correlation between the A/L ratio and biomarkers of low-grade chronic inflammation (Frühbeck et al., 2018). Overall, the current clinical and experimental data support interaction between inflammation and cardiac autonomic nervous system that facilitates both the initiation and perpetuation of cardiovascular disease (Pizzi et al., 2010;Williams et al., 2019). However, the potential relationships between the A/L ratio and measures of autonomic imbalance in new-onset paroxysmal AF have not been characterized in the literature. Therefore, in the present study, we investigated the associations between the A/L ratio and heart rate variability in patients with new-onset paroxysmal AF.

| Study population
This retrospective study included 85 consecutive patients with a first diagnosis of paroxysmal AF who were scheduled to receive radiofrequency ablation at the Renmin Hospital of Wuhan University from March 2019 to December 2020. A total of 85 matched individuals with normal sinus rhythm (NSR) were included as controls.
Paroxysmal AF was diagnosed according to the guidelines established by the European Society of Cardiology (Hindricks et al., 2021).
Patients with a history of any of the following clinical conditions were excluded, as these factors may affect the A/L ratio: previous paroxysmal AF, acute coronary syndrome, any structural heart disease, stroke, cardiac failure (left ventricular ejection fraction [LVEF] <50%), implantation of pacemaker, other arrhythmias (e.g., bradycardia, sick sinus syndrome, atrioventricular block, or ventricular arrhythmias), abnormal ≥5% effective rhythm according to 24-h long-range electrocardiographic examination, cancer, any autoimmune disease, any hematological disease, renal failure, any hepatic disease (e.g., nonalcoholic fatty liver disease or cirrhosis), thyroid insufficiency, any systemic acute disease, and any chronic infectious disease. All methods were performed in accordance with the relevant guidelines and regulations.

| Blood sampling and laboratory analyses
Venous blood samples were collected after fasting, centrifuged, and stored at −80°C until analysis. Samples for the measurement of fasting blood glucose, lipid levels, uric acid, N-terminal pro-B-type natriuretic peptide (NT-proBNP), high-sensitivity C-reactive protein (hs-CRP), and creatinine were analyzed by a Siemens Healthcare Diagnostics instrument. Plasma adiponectin and leptin levels were determined by enzyme-linked immunosorbent assay (ELISA) using kits from Millipore.

| Echocardiography
A transthoracic echocardiography exam with color Doppler echocardiography was performed for all participants by the same skilled ultrasound physician. The left ventricular end-diastolic diameter (LVEDD), and left atrial anteroposterior diameter (LAD) were measured using standard M-mode echocardiography. The left ventricle ejection fraction (LVEF) was calculated with the modified Simpson method.

| Holter monitoring and heart rate variability analysis
For all participants, 24-h long-range 12-channel electrocardiographic recordings (DMS300-4A, DM Software, Inc.) were obtained upon admission. To simulate actual clinical use and cardiac sympathetic activity throughout the day and night, no attempt was made to control patient or environmental factors. Therefore, we expect that continuous monitoring with 24-h long-range 12-channel electrocardiography provides information about shifting HRV throughout the day. All 24-h long-range electrocardiographic recordings were saved with a sampling frequency of 1 kHz (signal bandwidth 0.04-387 Hz) and a resolution of 1 µV/bit. After classifying the QRS morphology, the R-R intervals (longest and shortest) were confirmed manually until no QRS sequences were incorrectly labeled. Only sequences with normal QRS characteristics during 24 h (sinus rhythm) were analyzed for the HRV study. During analysis, patients with sinoatrial node dysfunction and a large number of abnormal rhythms (abnormal ≥5% effective rhythm) were excluded. The specific signal processing steps for computation of the HRV parameters are detailed elsewhere (El Aarbaoui et al., 2017). HRV, which reflects cardiac autonomic function, was analyzed using commercial software (H-Scribe Analysis System, Mortara Instrument, Inc.). The following parameters of HRV were calculated according to previously published methods and using customized and validated software (Circulation, 1996). HRV indexes included both time-domain parameters (i.e.,  transformation was used to convert the different successive RR intervals in the frequency domain. HF generally reflects cardiac parasympathetic nerve activity. Accordingly, the LF/HF ratio provides an indicator of the balance between sympathetic and parasympathetic activities, with higher LF/HF ratio values indicating increased sympathetic nerve excitability (Circulation, 1996). Analysis of the LF/HF ratio rather than either single component is widely accepted as a preferred technique for assessing cardiac autonomic nervous system balance and sympathetic activity (Xhyheri et al., 2012).

| Statistical analysis
All statistical analyses were carried out using SPSS 22.0 software (SPSS, Inc.). The included patients were assigned to two groups according to the median value of the A/L ratio among the study population, and comparisons were made between the groups with low and high A/L ratios. Continuous data are presented as mean ± standard deviation (SD) or median (interquartile range), and categorical data as number and percentage. The two-sample t test was used to compare variables that showed a normal distribution, and the Mann-Whitney U test was used to compare variables that showed non-normal distributions. Categorical data are reported as count (proportion) and were compared using the χ 2 test or Fisher's exact test. The Spearman correlation analysis was performed to evaluate the correlations between A/L ratio and indices of heart rate variability (i.e., LF, HF, and LF/HF ratio). Variables for which a significant correlation was found (p < .05) were included in the subsequent multivariate logistic regression analysis. For all analyses, statistical significance was recognized if p < .05.

| Clinical characteristics of patients with AF and controls with normal sinus rhythm
A total of 85 patients with new-onset paroxysmal AF and 85 people with NSR were included, and the baseline characteristics of the included participants are presented in Table 1. Significant differences were detected for NT-proBNP, LDL-cholesterol, total cholesterol, triglycerides, adiponectin, A/L ratio, LAD, LVEF, SDNN, rMSSD, PNN50, LF, HF, LF/HF ratio, and heart rate between the two groups.
Patients with new-onset paroxysmal AF were more likely to have lower values of LDL-cholesterol, total cholesterol, triglycerides, and LVEF and higher values of NT-proBNP, adiponectin, A/L ratio, LAD, SDNN, rMSSD, PNN50, LF, HF, LF/HF ratio and heart rate compared to those with NSR (all p < .05).

| Characteristics of patients according to A/L ratio
Overall, 85 patients with new-onset paroxysmal AF were included, and the characteristics of the included participants are presented in Table 2. Those with a higher A/L ratio (≥1.71, n = 43) were more likely to be male and to have a lower body mass index (BMI), triglyceride level, uric acid level, heart rate, hs-CRP level, LF, and LF/HF ratio but a higher HF compared with those with a lower A/L ratio (<1.71, n = 42; all p < .05). However, with the grouping of patients according to A/L ratios, no correlation was detected between adipokine levels and time-domain HRV indices. The patient groups with low and high A/L ratios showed no significant differences in the other observed characteristics.

| Association between leptin and LF
The results of Spearman correlation analysis showed that coronary heart disease, BMI, hs-CRP level, leptin concentration (Figure 1a), A/L ratio, LAD, LVEF, and heart rate were significantly correlated with LF (all p < .05, Table 3). Subsequent multivariate analysis also identified coronary heart disease, BMI, hs-CRP level, leptin concentration, and heart rate as independently associated with LF among all study participants (p < .05, Table 4).

| Association between adiponectin and HF
Univariate line regression analysis identified hs-CRP level, adiponectin concentration (Figure 1b), leptin concentration, A/L ratio, and Abbreviations: A/L, Adiponectin/Leptin; BMI, body mass index; CHD, coronary heart disease; HDL, high-density lipoprotein; HF, high frequency; hs-CRP, high-sensitivity C-reactive protein; LAD, left atrial diameter; LDL, low-density lipoprotein; LF, low frequency; LVEDD, left ventricular end-diastolic dimension; LVEF, left ventricle ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide; PNN50, percent of the number of times that the difference between adjacent normal R-R intervals >50 ms in the total number of NN intervals; rMSSD, root mean square successive difference of normal R-R intervals; SDANN, standard deviation average of NN intervals; SDNN, standard deviation of normal R-R intervals.
TA B L E 1 Clinical characteristics of patients with AF and controls with normal sinus rhythm heart rate as significantly associated with HF (p < .05, Table 5). Of these factors, only hs-CRP concentration and adiponectin concentration were independently associated with HF in the overall cohort according to the subsequent multivariate analysis (p < .05, Table 6).

| Association between A/L ratio and LF/ HF ratio
Univariate logistic regression analysis identified hypertension, gender, hs-CRP concentration, A/L ratio (Figure 1c), and LAD as factors significantly correlated with the LF/HF ratio (p < .05, Table 7). On the subsequent multivariate analysis, only hs-CRP concentration and A/L ratio were independently associated with the LF/HF ratio in the overall patient cohort (p < .05, Table 8).

| DISCUSS ION
In this case-control study of AF patients and NSR controls, we found that the serum adiponectin levels, A/L ratio, SDNN, rMSSD, PNN50, LF, HF, and LF/HF ratio were significantly higher in Note: Continuous variables are presented as mean ± SD or median and IQR; categorical variables are presented as number (percentage); values of p < .05 are indicated in bold.
Abbreviations: A/L, Adiponectin/Leptin; BMI, body mass index; CHD, coronary heart disease; HDL, high-density lipoprotein; HF, high frequency; hs-CRP, high-sensitivity C-reactive protein; LAD, left atrial diameter; LDL, low-density lipoprotein; LF, low frequency; LVEDD, left ventricular enddiastolic dimension; LVEF, left ventricle ejection fraction; NT-proBNP, N-terminal pro-brain natriuretic peptide; PNN50, percent of the number of times that the difference between adjacent normal R-R intervals >50 ms in the total number of NN intervals; rMSSD, root mean square successive difference of normal R-R intervals; SDANN, standard deviation average of NN intervals; SDNN, standard deviation of normal R-R intervals.
new-onset paroxysmal AF patients than in control individuals with NSR. Among the included patients with new-onset paroxysmal AF, we found that those with a higher A/L ratio had a higher HF

| Cardiac autonomic function and AF
Despite the availability of many studies on the subject, the pathogenesis of AF remains unclear. Its pathogenesis has been primarily attributed to mechanisms involving the cardiac autonomic function   (Annoura et al., 1999). However, further research is needed to evaluate the potential mechanisms underlying the association between hypolipidemia and AF.

| A/L ratio and heart rate variability
Obesity has consistently emerged as a risk factor for AF and the progression of paroxysmal to permanent AF, which is related to the release of adipokines by adipose tissue (Badimon et al., 2017).
Accordingly, dysfunction among adipose tissue as indicated by a low A/L ratio may represent a hallmark of obesity and metabolic syndrome, with increased levels of proinflammatory factors serving as potential mediators in the relevant pathophysiologic processes (Frühbeck et al., 2017). Therefore, it has been proposed that a low A/L ratio may be a novel and reliable surrogate biomarker of inflammation and a diagnostic factor for new-onset paroxysmal AF. Also, links between epicardial adipose tissue and AF were demonstrated in recent studies based on the interaction between adipose tissue and inflammation, further supporting our conclusions (Tran et al., 2019).
Indeed, previous studies showed significant association between the L/A (leptin/adiponectin) ratio and the severity of coronary lesions in patients with coronary artery disease patients according to the Gensini score (Rahmani et al., 2020). Similarly, use of the L/A ratio as a clinically useful biomarker was reported as a promising alternative for detecting early obesity-related metabolic disturbances of any kind (Larsen et al., 2018). Moreover, because the A/L ratio reflects the functionality of adipose tissue, this ratio may be a valuable index for identifying patients at risk for cardiometabolic diseases (Frühbeck et al., 2017). In the present study, the balance of cardiac autonomic tone was evaluated by applying power spectral analysis of heart rate variability, and the results provide a pathophysiological basis underlying the association between A/L ratio and cardiac autonomic tone.
Our previous study found that adiponectin acts on adiponectin receptor (Adipor) of cardiac ganglionated plexus (GP) neurons to activate the Adipor/AMPK/NF-κB signaling pathway, reducing the expression of proinflammatory factors, and preventing the occurrence of AF by inhibiting the activity of GP neurons, further confirming the protective effect of adiponectin in AF (Zhou et al., 2020). Taken together, our findings have further confirmed that an abnormal A/L ratio may serve as a novel index to predict the risk of new-onset paroxysmal AF or as a prognostic marker in AF. Further studies are needed to confirm these findings and expand our understanding of the potential role of the A/L ratio in risk stratification for new-onset paroxysmal AF patients.

| Adiponectin and heart rate variability
Previous studies have reported inconsistent results regarding the association between adiponectin level and AF risk (Macheret et al., 2015). Two cross-sectional studies showed that compared to the serum adiponectin level in individuals with sinus rhythm, those in patients with persistent AF are significantly higher (Shimano et al., 2008), while those in patients with paroxysmal AF are significantly lower (Choi et al., 2012). These conflicting results may be explained by the presence of a significantly larger pericardial fat volume in persistent AF patients compared with that in paroxysmal AF patients (Al Chekakie et al., 2010). Moreover, previous research indicated that a high plasma level of adiponectin is related to a smaller body size in elderly women, which corresponds to a larger left atrial volume index, and can serve as a predictive factor for AF recurrence after catheter ablation among patients with paroxysmal AF (Kim et al., 2018). Our results showed that the serum adiponectin level was independently and positively associated with HF power in patients with new-onset paroxysmal AF. Therefore, we speculate that adiponectin may be an active participant in the pathogenesis of new-onset paroxysmal AF.

| Leptin and heart rate variability
The mechanism of AF by which leptin regulates atrial muscle cell activity remains incompletely understood (Jenne & Tschopp, 1992;Jiang et al., 2020;Li & Liu, 2009;Lin et al., 2012Lin et al., , 2013. The literature related to the association of blood leptin level and heart rate variability parameters indicates a possible link between the leptin concentration and disturbances of the autonomic nervous system for some ethnicities (Pieterse et al., 2014). One study reported that hyperleptinemia may be related to cardiac autonomic dysfunction in patients with type 2 diabetes and visceral obesity (Kurajoh et al., 2015). In male patients with cardiac infarction, Piestrzeniewicz et al.
showed that leptin was positively associated with LF and the LF/ HF ratio, but negatively correlated with HF (Piestrzeniewicz et al., 2008). The results of the present study are inconsistent with those of previous studies in that leptin was only positively associated with LF in our patients with new-onset paroxysmal AF. Overall, the present study and those in the current literature illustrate the role of increased leptin secretion, which will contribute to autonomic imbalance. Therefore, the circulating adiponectin level may be a potential biomarker of AF or an independent predictor for recurrence of atrial arrhythmia after catheter ablation for AF. Potential interactions among these pathways are of potential significance and warrant further investigation.

| Study limitations
Several limitations of the present study should be noted. First, this was a retrospective observational study with a limited sample size, and the findings must be confirmed in prospective studies with larger samples. Second, it remains unclear whether an elevated A/L ratio is a cause or consequence of heart rate variability in patients with paroxysmal AF, and therefore, the associations between the A/L ratio and parameters of heart rate variability should be evaluated in future studies. Third, no control group of healthy people was included in the present study, and thus, we could not examine the differences between paroxysmal AF patients and a control group of healthy people. Finally, without longitudinal follow-up data, we could not assess the clinical impact of the A/L ratio and heart rate variability analysis on future events.

| CON CLUS ION
The results of the present study indicate that the A/L ratio, as well as the individual adiponectin and leptin levels correlated with cardiac autonomic function in new-onset paroxysmal AF patients. However, given the known limitations of HRV analysis during new-onset paroxysmal AF, the potential value of adipokine levels for the identification and evaluation of new-onset paroxysmal AF deserves further investigation.

ACK N OWLED G EM ENT
Thanks to everyone for supporting our final study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

AUTH O R S CO NTR I B UTI O N S
Conceived and designed the study: HJ. Data collection and analyzed the data: TZ and MW. Quality control the study and revision: MC, ZW, SW, and HH. Wrote the paper: TZ and MC. TZ, MC, and MW contributed to the work equally and should be regarded as co-first authors. Corresponding Author: KM and HJ. The manuscript was approved by all the above authors.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
Because this was a retrospective observational study and no treatment was tested in patients by the authors of this article, the Renmin Hospital of Wuhan University Ethics Committee granted an exemption from requiring ethics approval and waived the requirement to obtain informed consent from eligible patients. All methods were performed in accordance with the relevant guidelines and regulations.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the Renmin Hospital of Wuhan University. However, restrictions apply to the availability of these data, which were used under license for the current study, and accordingly, they are not publicly available. Data are, however, available from the authors upon reasonable request from the corresponding author Kezhong Ma (E-mail:

makezhong2020@163.com) and with permission of the Renmin
Hospital of Wuhan University.