Mechanisms Of Acute Proarrhythmic Effects Of Low Concentration BPA On Female Rat

BackgroundBishphenol A (BPA) was first synthesized by Aleksandr Dianin at 1891。It is the most important component of carbonate plastic, epoxy resins, and as a non-polymer additive to other plastics and rubber. Since 1950's, plastic manufacture developed very rapid, BPA is extensively used in consumer goods and products, such as baby bottles, beverage containers, dental sealants, and food cans. It was reported that annul BPA production had reach 6.4 billion pounds in 2003 and the related products in demand are still increasing. In an epidemiologic study performed in the U.S. BPA was detected in 95% of the testees in the urine, with an average urine concentration of about 10-9M。Therefore the effect of extensive BPA contamination on human health has attracted the researchers from many fields. It demonstrated that BPA as an endocrine disrupting chemical (EDC) of estrogen can imitate/antagonist the effect of estrogen. Under the acute situation, BPA can combine to estrogen receptor (ER)αand ERβ,initiate the rapid response. A lot of experimental evidence link BPA exposure to cancer, obesity, diabetes, and disorders of the reproductive, neuroendocrine and immune systems. And recently a cross-sectional study reported that high BPA levels were significantly associated with heart disease, including coronary heart disease (CHD), angina, and heart attack.According the literatures about BPA, we hypothesized that during acute situation low concentration BPA may promote arrhythmia in rat single cardiac myocyte and isolated heart. And as an EDC of estrogen, the effect of BPA on male and female may different. The molecular mechanisms of BPA may be that BPA combine the ER then activate the signal pathway associated with estrogen's rapid response. Meanwhile we also try to address the effects of BPA on pathological heart by observing the effects of BPA on IR arrhythmia induced by ischemia/reperfusion injury of in vitro whole heart.Following is the experiment design for proofing our hypothesis. First, we observed the dose-response curves of BPA by measuring contractility of female cardiac myocyte. Examined the acute effects of BPA or BPA+E2 on calcium spark, calcium transient, SR calcium content, L type calcium current, as well as after-contraction and after-transient of cardiac myocyte by photometry system, patch clamp and confocal microscopy, individually. we also observed that if the Ryanodine receptor (RyR) blocker can affect the proarrhythmic effect of BPA. Try to demonstrate the proarrhythmic effect of BPA on single cardiac myocyte and if it is sexual-specific and address its electrophysiological mechanisms.Second, the effects of BPA on the frequency of after-transient of ERβknockout transgenic mice cardiac myocyte which induced by rapid stimulation examined by confocal microscopy. Meanwhile contractility and after-transient of adult female rat cardiac myocyte after treated with agonists or antagonists of ERa or ERβalso been measured. Try to figure out what kinds of effect these two types of ER isoforms distribute to the arrhythmia response of cardiac myocyte to BPA and demonstrate the molecular mechanisms of BPA.The previous sections of this experiment are about the single cardiac myocyte. In the last section, we examined the effects of BPA on electrocardiogram (ECG) of in vitro whole heart. At the same time we also observed the effects of BPA on global IR injury arrhythmia and infarction size of pathological female rat isolated whole hearts. Try to address the effects of BPA on the electrophysiology of isolated whole heart. Therefore we demonstrated the proarrhythmic effects of low concentration BPA on female rat heart by combining the single cardiac myocyte with whole heart and normal heart with pathological heart. Section 1:The proarrhythmic effect of low concentration BPA on female rat cardiac myocytes and its electrophysiologic mechanismsObjectives:To observe the effect of BPA on contractility, calcium spark, calcium transient, SR calcium content, spontaneous after-contraction, after-transient of dissociated adult rat single cardiac myocytes. Investigate the proarrhythmic effects of BPA on single cardiac myocytes and if it has sexual difference. Try to address its electrophysiologic mechanisms.Methods:Use enzymes to dissociate female and male adult rat heart to single cardiac myocytes. Cells divided to four groups:control, BPA group, E2 group, BPA+E2 group. After exposure to each reagent two minutes, cardiac myocyte contractility and after-contraction were measured by video edge detection system, calcium transient and SR calcium content were examined by photometry system, and using confocal microscopy to test calcium spark and after-transient. L type calcium current was measured by whole cell patch clamp. After ryanodine receptors blocked by ryanodine, record the percentage of cells with spontaneous after-contraction induced by rapid stimulation.Results:The dose-response curve of BPA:Got the dose-response curve of BPA by measure contractility of female rat cardiac myocytes. The results show that dose-response curve of BPA was similar to E2 as a inverted " U " shape, The most efficacious concentration of BPA was 10-9M.The effects of BPA on the contractility of female and male cardiac myocytes: Both BPA and E2 increased the contractility of female rat cardiac myocytes. (p<0.01). Exposure to BPA and E2 at same time increased the contractility more (compares to control,p<0.01, compares to BPA or E2,p<0.01). As to the male cardiac myocytes, neither BPA nor E2 affected the contractility. nuclear receptors. Nonnuclear effect happened immediately after the receptors are activated, while genomic effects take longer to occur. BPA, as one of the estrogen EDCs, also can combine to ERs. This section using agonist or antagonist of ERa or ERβcombining with ERβknockout transgenic mice model to demonstrate how ERs mediate the proarrhythmic effects of BPA. Data show that activating ERa inhibits the contractility of female adult rat cardiac myocytes and the DADs promotion effects of BPA+E2. When using MPP to block the ERa, BPA or BPA+E2 increases the contractility of female adult rat cardiac myocyte, increases the incidence rate of DADs. Activating ERβenhances the contractility of female rat cardiac myocytes, induces DADs. Blocking ERβcan decreases the contractility of female rat cardiac myocytes, inhibits DADs induced by BPA+E2. As to the male cardiac myocytes, activating ERαinhibits the contractility, activating ERβincrease contractility and the incidence rate of after-transient. results of ERβknockout transgenic mice show that BPA+E2 can not increase contractility and incidence rate of DADs, even the ERβagonist. All these evidences demonstrate that the proarrhythmic effects of BPA mediated by ERβ. However, ERa mediate negative effects. These two previous sections discuss about the single cardiac myocytes, because of the complexity of whole heart electrophysiology, it is quite different between the single cardiac myocyte and whole heart. Next section we will try to investigate the effects of BPA on the arrhythmogenesis of whole heart. Section 3:The acute effect of low concentration BPA on ECG of isolated whole heart and arrhythmia of ischemia/reperfusion injury.Objective:To investigates the effects of BPA+E2 on ECG of in vitro isolated whole heart, under the normal situation or given isoproterenol (ISO). Meanwhile observe the effects of BPA on IR injury arrhythmia. Try to demonstrate the proarrhythmic effects of BPA on whole heart.Methods:In vitro isolated whole heart divided into four groups, control, BPA+E2, ISO and BPA+E2+ISO. The heart of female and male adult rat removed quickly, mounted on Langendorff and perfused to steady state, recorded ECG. As to the ischemia/reperfusion model, female adult rat isolated heart mounted on Langendorff and perfused to steady state, stopped perfusion,20 minutes later, reperfused with normal solution or BPA+E2 solution, ECG recorded synchronously. One hour later, took off heart from Langandorff, stained by TTC, calculated infarction size by imageJ.Results:The effects of BPA+E2 on ECG of female rat isolated heart:Under normal situation, ventricular arrhythmia didn't happen in whole heart. Even given BPA+E2, the incidence rate was still very low (0 vs 1.22±0.72/20min p>0.05). Ventricular arrhythmia occurred occasionally in ISO group, but there were no significant difference between control and ISO (0 vs 1.60±1.10/20min,p>0.05). However BPA+E2+ISO dramatically increased the frequency of ventricular premature beats to 6.72±2.59/20min (compare to control, BPA+E2, ISO,p<0.05).The effect of BPA+E2 on the ECG of male rat isolated heart:Similar to the result of female rat isolated heart, exposure to BPA+E2 did not induce ventricular arrhythmia, the frequency of ventricular premature beat is 0/20min. However under the stress situation mimicked by ISO, the frequency of ventricular premature beat didn't increase by exposure to BPA.+E2The result of IR arrhythmia:the ventricular arrhythmia of control stopped 5 minutes after reperfusion.10 minutes later it almost recovered to normal. As to BPA+E2, it still has ventricular arrhythmia (VF) at two minutes after reperfusion, five minutes later, it changed to ventricular tachycardia (VT), Until ten minutes ventricular arrhythmia stopped. the lasting time of IR injury arrhythmia of control was 280.10±76.62sec, BPA+E2 prolonged the ventricular arrhythmia lasting time to 529.70±89.91sec (compare to control, p<0.05), and increase the percentage of VF (30.49±8.06% vs 9.98±5.02%, p<0.05), therefore, BPA+E2 not only prolong the lasting time of IR injury arrhythmia, but also deteriorate the arrhythmia.The effect of BPA+E2 on infarction size of IR injury:BPA+E2 can dramatically decreased the infarction size of female rat IR injury heart (p<0.05).Conclusions and DiscussionsPrevious sections showed that BPA can promote DADs of single cardiac myocytes, and demonstrated the elctrophysiological mechanisms and molecular mechanisms. There are three types of arrhythmia, first is the automaticity of cardiac myocyte increasing. Second is reentry arrhythmia. The third one is triggered activity which includes early after depolarization and DAD. It shows as a ventricular arrhythmia such as ventricular premature beats, VT, and VF on ECG. This section examined the effects of BPA on ECG of in vitro isolated heart. Results showed that BPA combined with E2 can not induced ventricular arrhythmia. However, under the stress situation mimicked by giving ISO, BPA+E2 can dramatically increased the frequency of ventricular premature beats of female isolated whole heart. As to the male in vitro isolated heart, there are no different between ISO group and BPA+E2+ISO group. Therefore, we observed the effects of BPA on female rat IR injury pathological isolated heart. Results showed that BPA+E2 can increase the lasting time of reperfusion arrhythmia significantly. Enhance the percentage of VF, deteriorate ventricular arrhythmia. As to the infarction area, BPA+E2 can decrease the infarction size which is consistent with published literatures.According the results of these three sections, we concluded that as an EDC, BPA can combine to the membrane ERβ,activate series of signal pathways, enhance the frequency of cardiac myocyte calcium spark by increasing the RyRs opening and SR calcium content, then induced delayed afterdepolarization, promote cardiac ventricular arrhythmia, especially under pathological situation. And these effects are female-specific. However, the effects of BPA mediated by ERαare negative.