The risk of developing cardiovascular diseases, including cardiac arrhythmias, increases considerably with advancing age1. Atrial fibrillation (AF) is the most common sustained heart rhythm disorder, affecting between 2% and 4% of the world population according to recent data2. The continuous increase in AF prevalence has led to predictions about the magnitude of the AF burden in the future. Krijthe et al. anticipated that in the European population the prevalence of AF will increase to 2.5% by 2030 and to 3.5% by 20603. Even though the world population is progressively older4 and the prevalence of numerous factors known to be involved in AF onset and maintenance is on the rise5,6, these factors can only partially explain the continuous increase in AF prevalence recorded over the past decades, suggesting that other, yet unidentified factors may also contribute to this worrisome epidemiologic progress7.
Bisphenol A (BPA), one of the most extensively used chemical compounds, is widely utilized in the production of epoxy resins and polycarbonate plastics. Since these materials are present in a vast variety of common products, including in plastic bottles, food containers and cans, dental materials, water pipes, toys, protective equipment, electronics, and even in numerous medical devices, human exposure to BPA via different routes (i.e., oral, cutaneous, or by inhalation) is both important and continuous8. In addition, the presence of BPA monomers that remain unbound during the production process makes these BPA molecules easily transferable to the human body even during common exposure9. Studies indicated the presence of BPA in the urine of more than 90% of the general population, with higher BPA concentrations in workers in the plastics industry10.
The main health implications of BPA derive from its role as an estrogen endocrine disruptor. Accumulating data from both human11 and animal12 studies incriminate BPA exposure as an important contributor to a vast variety of diseases, including diabetes mellitus, obesity, and various malignancies. Studies have also suggested a potential link between BPA exposure and cardiovascular diseases such as arterial hypertension, ischemic heart disease, and ventricular arrhytmias11. However, no study has assessed to date the potential impact of BPA exposure on atrial arrhythmogenicity. Moreover, even the BPA-related cardiotoxicity remains a matter of debate. Although several cross-sectional clinical studies support a relationship between BPA exposure and cardiovascular disease10,13, such studies cannot ascribe causation, whereas preclinical studies have mostly focused on
Accordingly, we aimed to evaluate the effects of chronic exposure to both high BPA concentrations and BPA concentrations relevant for common human exposure on atrial electrophysiological parameters, on cardiac autonomic modulation, and on atrial spontaneous and electrically-triggered arrhythmogenicity in healthy adult rats.
22 adult female Wistar rats (263 ± 12.54 g) were randomly divided into three groups: Control (n = 7), BPA (n = 7), and hBPA (n = 8). Animals in the BPA group were exposed to BPA doses of 50 μg/kg of body weight/day, considered, according to previous studies15, relevant for common human exposure16. In accordance with previous studies17, animals in the high BPA (hBPA) group were administered 25 mg/kg of body weight of BPA/day. Bisphenol A (Sigma-Aldrich, St. Louis, MO) was administered in all rats in the drinking water for 9 consecutive weeks. Control rats received normal tap water throughout the study. All protocols used in the present study complied with the International Council for Laboratory Animal Science guidelines (Directive 2010/63/EU) and were approved by the local Ethics Committee.
Body weight was determined weekly for all rats throughout the study. Systolic blood pressure (SBP) was assessed in all rats by photoplethysmography. Briefly, a pneumatic cuff was placed proximally on the rats’ tail and was inflated/deflated using a programmable electro-sphygmomanometer (PE-300; Narco Bio-Systems, Austin, TX). The photoplethysmography sensor was placed distally, at the level of the caudal artery. The cuff and photo-transducer signals were routed to a computer equipped with an acquisition board. Signals were recorded and analyzed using a program developed using the LabVIEW 8.6 software (National Instruments, Austin, TX). Each animal underwent SBP measurement after eight weeks of BPA or normal water exposure.
After six weeks of BPA or normal water administration, all animals were implanted under isoflurane inhalation anesthesia (4 L/min, 2.5%) with an electrocardiographic radiotelemetry transmitter (Data Science International, St. Paul, MN). The transmitter was placed in a dorsal, subcutaneous pocket, and the two ECG leads were tunneled subcutaneously and secured in a lead II configuration. Continuous 24-h ECG monitoring was performed in freely moving rats and all ECG traces were analyzed using a dedicated software.
The mean heart rate and the occurrence and duration of spontaneous atrial arrhythmic events (i.e., number of atrial premature beats/24-h, and number and duration of AF episodes/24-h) were assessed at baseline and after application of an atrial electrical stimulation protocol, as described below. Atrial premature beats were defined as early P waves, with morphology different than the sinus rhythm P waves, followed by narrow QRS complexes. Spontaneous AF was defined as the occurrence of three or more consecutive irregular, supraventricular beats (i.e., irregular ventricular response with narrow QRS complexes), in the absence of clearly identifiable P waves.
Continuous ECG recordings obtained prior to atrial electrical stimulation were also used for heart rate variability (HRV) analysis in both the time and frequency domains. The HRV parameters were determined by analyzing beat-to-beat variations in RR intervals, as described previously.18 The standard deviation of normal RR intervals (SDNN), the root mean square of the successive RR interval differences (RMSSD), and the percentage of successive RR intervals that differed by more than 5 ms (pNN5) were assessed as time domain parameters, whereas the low-frequency (LF; 0.3–0.6 Hz) and the high-frequency (HF; 0.6–2.5 Hz) components of the HRV spectrum and the LF/HF ratio were assessed as frequency domain parameters.
Atrial fibrillation inducibility was assessed
At 24 h after the stimulation protocol, the animals were euthanized, the hearts were rapidly excised, and placed in oxygenated Krebs-Henseleit solution (NaCl 118.00 mM, KCl 4.70 mM, NaHCO3 25.00 mM, MgSO4 1.20 mM, CaCl2 1.25 mM, KH2PO4 1.20 mM, and glucose 11.00 mM). The left atrium was isolated and transferred into the Steiert organ bath, where it was continuously perfused with oxygenated Krebs-Henseleit solution at 37°C. The atrial tissue was electrically stimulated at a frequency of 1 Hz and the transmembrane atrial action potentials were recorded using a microelectrode filled with 3 M KCl (resistance 2–4 MΩ). Atrial action potentials were recorded at baseline and after exposure to proarrhythmogenic stimuli (i.e., 10−5 M adrenaline solution, 10−5 M acetylcholine solution, and 1.25 mM CaCl2 solution), with 15-min washing intervals between the different solutions. For each stage of the protocol, 10 action potentials were analyzed, and the depolarization rate and the action potential duration until reaching 50% (APD50) and 90% (APD90) of complete repolarization were assessed, as described previously20.
All statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software; San Diego, CA). Given the low sample size, all between-group comparisons were performed using the non-parametric Mann-Whitney test. Multiple comparisons were performed using Kruskal-Wallis test. The results are reported as median and interquartile range. The p value was set at 0.05 for statistical significance.
At the end of the study, both groups exposed to BPA showed lower body weight than the Control rats (both p = 0.04), and this effect was not affected by the BPA dose (Figure 1). No significant difference was found in either the heart rate or the SBP values between the Control and the BPA, nor between the Control and the hBPA groups (all p >0.05; Table 1).
Body weight in Control rats and in rats exposed to usual (BPA) and high (hBPA) bisphenol A doses for 9 weeks. Data are expressed as median and interquartile range; p-values were calculated using the Mann-Whitney test.
Systolic blood pressure and mean 24-h heart rate in Control rats and in rats exposed to usual (BPA) and high (hBPA) bisphenol A doses for 9 weeks
SBP (mmHg) | 105.0 (87.5–117.5) | 110.0 (100.0–127.5) | 110.0 (110.0–120.0) | 0.46 |
HR (bpm) | 362.6 (348.4–406.5) | 364.1 (354.9–375.6) | 352.4 (347.1–364.6) | 0.55 |
The values are expressed as median and interquartile range; p-values refer to between-group comparisons based on the Kruskal-Wallis test.
HR – heart rate; SBP – systolic blood pressure
Heart rate variability analysis revealed significantly higher HF (p = 0.001) and LF (p = 0.04) components of the HRV spectrum in the BPA compared to the Control rats, while no significant difference was observed in the LF/HF ratio (p = 0.71), nor in the time domain HRV parameters (all p >0.05) between the two groups (Table 2). Meanwhile, rats in the hBPA group presented significantly higher RMSSD, pNN5, and HF components of the HRV spectrum, reflecting higher parasympathetic activity, compared to the Control rats (all p< 0.05; Table 2). In addition, the LF/HF ratio was significantly lower in the hBPA compared to the Control rats (p = 0.02), reflecting a shift of the autonomic activity toward vagal dominance in the hBPA rats (Table 2).
Heart rate variability parameters in Control rats and in rats exposed to usual (BPA) and high (hBPA) bisphenol A doses for 9 weeks
SDNN (ms) | 21.12 (15.71–22.03) | 20.66 (18.93–26.77) | 23.54 (21.16–28.11) | 0.46 | 0.10 |
RMSSD (ms) | 3.43 (3.14–4.16) | 4.28 (4.13–4.71) | 4.37 (4.93–5.54) | 0.16 | 0.03 |
pNN5 (%) | 11 (11–20) | 19 (19–22) | 22 (17–31) | 0.14 | 0.04 |
LF (ms2) | 1.70 (1.68–1.91) | 2.76 (2.06–3.12) | 3.01 (1.21–4.73) | 0.04 | 0.63 |
HF (ms2) | 5.37 (4.69–5.94) | 9.06 (7.89–9.97) | 9.22 (7.60–11.68) | 0.001 | 0.001 |
LF/HF | 0.36 (0.28–0.38) | 0.35 (0.25–0.37) | 0.24 (0.17–0.29) | 0.71 | 0.02 |
The values are expressed as median and interquartile range; p-values refer to between-group comparisons based on the Mann-Whitney test.
HF – high-frequency (0.6–2.5 Hz) signals; LF – low-frequency (0.3–0.6 Hz) signals; LF/HF – the ratio of low- to high-frequency components; pNN5 – percentage of successive RR intervals that differed by >5 ms; RMSSD – root mean square of successive RR-interval differences; SDNN – standard deviation of normal RR intervals
Regardless of the dose, BPA exposure had no significant effect on baseline action potential parameters (Table 3); i.e., the depolarization rate, APD50, and APD90 were all similar between the BPA and the hBPA groups, and the Control group, respectively (all p >0.05). Action potential changes in response to adrenaline, acetylcholine, and Ca2+ overload were also similar between the BPA and the hBPA groups, and the Control group (all p >0.05).
Atrial action potential parameters at baseline in Control rats and in rats exposed to usual (BPA) and high (hBPA) bisphenol A doses for 9 weeks
Depolarization rate (mV/s) | 26.2 (19.1–41.6) | 26.0 (18.7–40.0) | 21.8 (15.3–26.3) | 0.39 |
APD50 (ms) | 14.0 (12.9–19.9) | 14.4 (13.9–16.7) | 17.4 (15.6–23.3) | 0.10 |
APD90 (ms) | 37.4 (35.2–43.6) | 37.9 (32.5–43.2) | 43.4 (42.6–52.5) | 0.16 |
The values are expressed as median and interquartile range; p-values refer to between-group comparisons based on the Kruskal-Wallis test.
APD50 – action potential duration until reaching 50% of complete repolarization; APD90 – action potential duration until reaching 90% of complete repolarization
The occurrence and the number of electrically-induced AF episodes was not significantly different between the BPA and the hBPA groups, and the Control group, respectively (all p >0.05). Also, there was no significant difference in the number of atrial ectopic beats or in the number and duration of spontaneous AF episodes either at baseline or during the post-electrical stimulation period between the Control and the BPA groups (all p >0.05; Figure 2). However, hBPA rats presented a significantly higher number of atrial ectopic beats (p = 0.04), but not of spontaneous AF episodes (p = 0.60) at baseline compared with the Control rats (Figure 2). Moreover, during the period following the
Spontaneous atrial arrhythmogenicity at baseline and after transesophageal atrial burst pacing in Control and in rats exposed to usual (BPA) and high (hBPA) bisphenol A doses for 9 weeks. The figure illustrates the number of atrial premature beats (A and D), the number (B and E) and the duration (C and F) of spontaneous atrial fibrillation (AF) episodes per 24-h at baseline (upper panels – Baseline) and during the period following the
In the present study, BPA exposure was associated with significantly lower body weight, independently of the dose. To date, several clinical epidemiological studies indicated an association between BPA exposure and weight gain21,22,23. However, although all those studies showed a link between urinary BPA levels and obesity, neither of them was able to establish a cause-effect relationship. Contrarily, in a prospective study conducted on elderly individuals, the authors found no significant association between BPA serum concentrations and fat mass or distribution assessed by imaging techniques24. Meanwhile, early prepubertal exposure of female mice to high doses of BPA was associated with lower body weights compared with control, but no effect of BPA on body weight was observed during adulthood25. The lower body weight gain related to chronic exposure to BPA observed in the present study in healthy adult female rats is most likely the consequence of β-estrogen receptors activation leading to alterations in adipose tissue metabolism and increase in energy consumption26.
The data in the literature regarding the effect of BPA on blood pressure are also highly controversial. While several epidemiological studies showed an association between BPA exposure and high blood pressure27,28,29, others showed, on the contrary, a negative correlation between the two30. Belcher et al.31 showed a significant decrease in blood pressure following BPA exposure in CD-1 mice, independently of gender, whereas in their
Even though several clinical studies investigated the link between BPA exposure and cardiovascular diseases, little is known to date regarding the mechanisms that could underlie the potential cardiovascular toxicity of this extensively used chemical compound. It has been suggested that the potential toxic effects of BPA could be related to its function as an endocrine disruptor33. The protective role of endogenous estrogens on the cardiovascular system is well known, but the effects of exogenous estrogen administration remain highly controversial34.
Data regarding the cardiovascular toxicity of BPA are extremely limited. Several epidemiological studies demonstrated significant associations between BPA exposure and various cardiovascular diseases. None of those studies could establish, however, a causal BPA-cardiovascular disease relationship. Meanwhile, experimental studies, most of them conducted
The increasing prevalence of AF2 in the past decades raises serious questions about a potential atrial proarrhythmogenic effect of environmental factors, including BPA. The mechanisms that underlie AF onset and persistence are complex and remain incompletely elucidated. Broadly, these mechanisms rely on ectopic activity and re-entry35, occurring as a result of electrical remodeling, modulated by changes in various ion currents, structural remodeling, via fibrosis, and autonomic remodeling, expressed as an alteration in sympathetic and/or vagal tone36,37,38.
In previous experimental studies, acute exposure to high doses of BPA was associated with ventricular proarrhythmic effects. In female rodents, BPA exposure increased ventricular arrhythmogenicity in both
Additionally, exposure to BPA, regardless of the dose, has been shown to cause morphological changes of the heart. Several studies reported increased myocardial surface area after exposure to various BPA doses, related to either impaired mitochondrial function or up-regulation of genes involved in cardiac hypertrophy42,43,44. High-dose BPA was also shown to trigger myocardial estrogen receptor activation-mediated fibroblast proliferation, collagen production, and consequent fibrosis, and to alter cardiac mechanical function44,45, which could all contribute to increased cardiac arrhythmogenicity.
To the best of our knowledge, this is the first study designed to evaluate the effect of BPA exposure on atrial arrhythmogenicity. In the present study, exposure to BPA doses relevant for human exposure did not affect atrial arrhythmogenicity. Meanwhile, in baseline conditions, high BPA doses significantly increased the number of atrial premature beats, but not of spontaneous AF episodes. Although BPA exposure did not seem to affect AF inducibility during transesophageal atrial pacing, high BPA doses induced a significant atrial proarrhythmic effect following electrical stimulation of the atria. A 25 mg/kg/day BPA dose led to a significant increase in the number of atrial premature beats/24-h, as well as in the number and duration of post-electrical stimulation spontaneous AF episodes. These findings suggest that BPA exerts a dose-dependent effect on atrial arrhythmogenicity, with high BPA doses exhibiting not only ventricular14,15, but also atrial proarrhythmic effects. Our findings therefore suggest that massive exposure to BPA, such as that occurring in workers in the plastics industry, may lead to important atrial proarrhythmic effects. In healthy adult female rats, exposure to human-relevant doses did not appear to produce similar effects. However, it remains to be established whether the same is true in the presence of underlying cardiovascular disease.
The exact mechanisms underlying high-dose BPA-related atrial arrhythmogenicity remain to be established. According to our
To the best of our knowledge, this is the first study designed to investigate, both
The present study demonstrates that chronic exposure to high doses of BPA exhibits significant atrial proarrhythmic effects in healthy adult female rats, suggesting that important accidental or occupational BPA exposure could lead to serious cardiovascular effects, including in healthy individuals. Our data indicate BPA-induced vagal hyperactivation as a potential mechanism underlying this BPA-related atrial proarrhythmic effect. Exposure to BPA doses relevant for usual human exposure did not have a similar effect. However, future studies remain to establish whether this is also true in the presence of underlying cardiovascular disease.