Endogenous estrogens are well recognized to play important roles in the regulation and maintenance of sex-specific differences in cardiac physiology and pathophysiology; however, little is known about the cardiac effects of environmental estrogenic EDCs, such as the ubiquitous BPA, and their interactions with endogenous estrogens. In the present study, we show that physiologically-relevant concentrations of BPA and E2 have female-specific pro-arrhythmic effects in rodent cardiac myocytes and whole hearts. The effects of BPA and E2 are mediated by ERβ-signaling, through rapid alteration of myocyte Ca2+ handling, particularly by increase in Ca2+ leak from the SR. Our animal study results, for the first time, suggest a potential contributing role of BPA (and potentially other estrogenic EDCs) in the development of cardiac arrhythmias in the female heart.
When assessed using representative urine samples, BPA was detected in over 90% of the US population with mean concentration in the low ng/mL, or low-nanomolar range 
. As an indication of internal exposure, unconjugated BPA in plasma, serum or blood was detected in most individuals in various sampled populations, with mean concentration also in the low-nanomolar range 
. Initial dose-response analysis of the rapid effects of BPA and E2 in female myocytes was performed using myocyte contractility as an index. For each compound, the dose-response curve had an inverted-U shape; a stimulatory effect on contractility was observed at doses as low as 10−12
M, with 10−9
M being the most efficacious (data not shown). Therefore, based on experimental considerations, estimated human exposure levels, and BPA's pharmacodynamic profile, 10−9
M was selected for further analysis. We demonstrated here that in ventricular myocytes from female but not male rats, exposure to 1 nM BPA or E2, and particularly BPA combined with E2, markedly increased the percentage of myocytes with spontaneous excitations following repeated pacing. These triggered activities are indicative of delayed after-depolarizations, or DADs, a myocyte phenomenon that is recognized as a key non-reentrant arrhythmogenic mechanism 
. Delayed after-depolarizations are generated under SR Ca2+
overload condition and are the result of diastolic spontaneous Ca2+
release from the SR. Such spontaneous SR Ca2+
release triggers Ca2+
extrusion by the Na+
exchanger (NCX); the activity of the electrogenic NCX generates a depolarizing current, leading to excitation of the myocyte during the diastole. These ectopic excitations can propagate through the myocardium and initiate arrhythmia events 
. Key factors in the development of DADs include increased SR Ca2+
load, and abnormal SR Ca2+
release (i.e., SR Ca2+
leak). In particular, aberrant RyR opening and diastolic SR leak have been shown to be a central factor in the development of DADs and lethal ventricular arrhythmias under disease conditions such as heart failure 
. Consistent with the mechanism of DAD development, we found that in female rat myocytes, rapid exposure to BPA or E2 markedly increased SR Ca2+
reuptake, SR load, and the fraction of SR Ca2+
release on a beat-to-beat basis. Further, E2 and BPA significantly increased the frequency of spontaneous Ca2+
sparks, likely as a result of increasing RyR open probability. Supporting a key role of abnormal SR leak in the pro-arrhythmic effect of BPA in female myocytes, we showed that suppression of RyR release by ryanodine, while not affecting normal Ca2+
transients, abolished DADs triggered by BPA alone or BPA combined with E2.
Unlike the robust response of female myocytes to BPA and E2, neither compound induced arrhythmias under baseline conditions. However, with β-adrenergic receptor stimulation, BPA combined with E2 resulted in a marked increase in ventricular arrhythmic events, including premature ventricular contractions and ventricular tachycardia in female rat hearts. The discrepancy between the effects of estrogens at the myocyte and whole heart levels likely reflects the fact that only a fraction of the single myocyte-level abnormal electrical activities may propagate and result in arrhythmic events. How myocyte-level triggered activities lead to whole-myocardium arrhythmias, particularly sustained arrhythmias, is complex and not fully understood. Catecholamines affect myocyte mechanics by enhancing Ca2+
influx through the L-type Ca2+
channels, SR reuptake (and consequently SR Ca2+
load), and RyR activities 
. These effects favor SR overload and abnormal SR Ca2+
release, and likely potentiate the pro-arrhythmic actions of these estrogens. Our results suggest that BPA exposure may become a particularly significant factor for arrhythmogenesis in females under stress conditions, and possibly target females with existing cardiac pathophysiological conditions that provide the substrate for arrhythmogenesis.
The rapid effects of BPA on DAD development and Ca2+
handling appeared to be amplified in the presence of E2. This intriguing experimental effect raises the possibility that the pro-arrhythmic effect of BPA may be more pronounced in females with higher levels of endogenous estrogens, such as during pregnancy. Bisphenol A has been shown to have additive or synergistic effects with E2 or xenoestrogens in various cell types or systems in other studies 
. These results contrast with the findings in rat cerebellum, where BPA alone mimics the rapid effects of E2, but acts as an antagonist when combined with E2 
. The mechanism(s) underlying the interaction between BPA and E2 is currently unknown. The amplified effect of BPA and E2 in myocytes cannot be mimicked by doubling the dose of BPA or E2 (), suggesting that BPA elicits rapid effects via a mechanism that more complex than can be explained by considering E2 and BPA as equivalent rapid signaling estrogens. Analyses of the actions of BPA and E2 in female myocytes indicate that these effects are mediated via differential intracellular signaling through ERα and ERβ receptor (unpublished results). The known difference in affinity and binding properties of BPA and E2, and the differences in their signaling effects could account for the complex actions of E2 and BPA response. The interaction of BPA and E2-mediated effects observed here support further the notion that estrogenic actions are diverse and tissue and sex-specific; the endocrine disruption actions of estrogenic EDC should be assessed in the context of endogenous estrogens and possibly higher order mixtures of other estrogenic EDCs.
Bisphenol A elicits its biological responses through activation of ER or ER-like receptors. Both ERα and ERβ, including those localized to the membrane, have been shown to be expressed in cardiomyocytes from species including rat and human 
. Our results using the ERβ-/- mouse model strongly suggest that ERβ-mediated signaling plays a central role in the rapid actions of BPA and estrogen in female rodent cardiac myocytes. Consistent with this notion, we found that in female rat myocytes, the ERβ agonist DPN mimicked the rapid effects of BPA and E2, and the ERβ selective antagonist PHTPP (but not the ERα antagonist MPP) abolished the rapid effects of estrogens; G1, an agonist of the orphan G-protein coupled receptor GPR30, had no detectable effect in female myocytes (data not shown). A similar key role of ERβ in mediating the rapid effects of estrogenic EDCs has been described in cerebellar granular cells 
. Membrane ERs have been shown to activate kinases including protein kinase A and C 
, which are known to modulate various elements involved in myocyte Ca2+
and may play important roles in mediating the rapid action of BPA and E2 in female rodent myocytes.
Despite the perception that female hormones protect women from cardiovascular disease, disease of the cardiovascular system is the leading cause of mortality for women in the US. According to the American Heart Associate statistics, since 1984 more women than men have died of cardiovascular disease every year in the US; 53% of total cardiovascular disease deaths occur in women. There is well-recognized sexually dimorphism in cardiovascular disease, including cardiac arrhythmias. Compared with men, women have a lower incidence of sudden cardiac death owning to the protective effect of estrogens on coronary artery disease, and have lower risk of atrial fibrillation; however, women have higher rates of long QT syndrome, sudden cardiac death in the absence of coronary artery disease, and ventricular arrhythmias post myocardial infarction 
. Incidence of arrhythmias increases during pregnancy, and in women taking oral contraceptives 
, suggesting that female sex hormones may contribute to arrhythmogenesis. Our study provides the first experimental evidence suggesting that exposure to estrogenic EDCs like BPA, and the unique sensitivity of female hearts to estrogens, may play a role in arrhythmogenesis in the female heart. Elucidation of the cardiac effects of endogenous estrogen, environmental estrogenic EDCs, and their interactions is important for assessing the unique cardiovascular benefits and/or risks of both sexes, and may facilitate the development of therapeutic measures that protect against the cardiac risks associated with estrogenic EDC exposure.