低剂量BPA对雌性大鼠的急性促心律失常作用及其发生机制
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摘要
研究背景与总体思路
1891年Aleksandr Dianin第一次人工合成了化合物双酚A (Bisphenol A,BPA)。从此揭开了人类塑料工业的新篇章。BPA是聚碳酸酯塑料及树脂的主要成分之一,五十年代以来,随着塑料业的快速发展,BPA越来越广泛地应用于人类日常生活用品中,例如塑料婴儿奶瓶,饮料瓶,牙齿封闭剂及塑料食物盒等,且人类对BPA的需求越来越高,其对人类环境的污染也日益严重,从而使得越来越多的人群接触BPA,大规模的人群调查研究发现95%以上的人群尿液中能检测出BPA,其平均浓度为10-9M左右。因此由于BPA的广泛污染而给人类带来的影响也成为各个领域的研究者高度关注的话题。目前已证实BPA能.模拟／拮抗雌激素的作用,而被认为是一种雌激素的内分泌干扰化合物。作为雌激素的内分泌干扰化合物,BPA能结合雌激素受体(Estrogen Receptor, ER)α和β。发挥由ER介导的急性反应。有研究发现BPA具有促肿瘤,糖尿病和肥胖等发生发展作用,对机体的内分泌,生殖系统也能产生影响,而且认为人体尿液中的BPA浓度与心血管事件之间有着一定的相关性。
根据目前的研究现状与结果,我们试图证实低剂量BPA在急性状态下对大鼠单个心肌细胞及离体心脏可能具有促心律失常作用。且由于BPA为性激素内分泌干扰物,因此其对雌雄性大鼠可能发挥着不同的作用。这一作用可能是通过结合于细胞膜上的ER,快速激活相关的信号通道而实现的。此外,本研究还试图探讨在心脏缺氧／再灌注损伤时,BPA对灌注后心律失常的影响。
为了证实这一推测。本研究方案如下：
首先,以体外分离的成年大鼠心室肌细胞为研究对象,以心肌细胞收缩率为指标,观察BPA对不同性别大鼠心肌细胞的作用有何不同,同时在急性状态下单纯给予BPA或在有雌二醇(Estrodial, E2)存在时给予BPA,用离子成像系统和共聚焦显微镜等手段检测心肌细胞钙火花,钙瞬变,肌浆网钙负荷以及延迟后除极相关指标after-contraction和after-transient, L型钙电流等钙动力学相关指标的变化,并观察兰尼定受体阻断剂兰尼定是否影响BPA的作用,初步探讨BPA对单个心肌细胞的促心律失常作用及其是否具有性别特异性,并探讨其发生的电生理作用机制。
其次,以ERβ基因敲除小鼠为实验模型,观察BPA对心肌细胞快速刺激后产生after-transient的影响。同时检测ERα和β兴奋剂对成年雌性大鼠心肌细胞收缩率及after-transient的作用,并在给予BPA的同时给予ERα和β拮抗剂,观察二者是否影响BPA的作用,从而试图解释ER的两个亚型α和β如何介导在BPA的急性促心律失常作用,初步探讨BPA的分子作用机制。
最后,以体外灌注的整体心脏作为研究对象,检测BPA对其心电图的作用,同时观察缺氧／再灌注损伤时,BPA对缺氧／再灌注损伤室性心律失常及梗死面积的影响,阐述BPA对离体整体心脏电生理的影响。从而以单个细胞结合整体心脏,正常生理状态结合病理状态,全面地探讨低剂量BPA对雌性大鼠心脏的促心律失常作用。
第一部分：低剂量BPA对雌性大鼠心肌细胞的急性促心律失常作用及其电生理机制
目的：
以体外分离的成年大鼠心肌细胞为研究对象,通过离子成像系统,共聚焦显微镜及膜片钳等检测手段观察心肌细胞收缩率,心肌细胞钙火花,钙瞬变,肌浆网钙负荷以及延迟后除极指标after-contraction和after-transient, L型钙电流等钙动力学相关指标的变化,初步探讨BPA促进单个心肌细胞心律失常发生的电生理机制及其性别特异性。
方法：
雌雄性成年大鼠以酶解法分离出单个的心室肌细胞,再按实验要求分为四组,正常对照组,BPA组,E2组,BPA+E2组,各组细胞分别经药物处理2分钟后,用Video edge detection system测量心肌细胞收缩率及after-contraction,用离子成像系统测定心肌细胞钙瞬变,肌浆网钙负荷,用共聚焦显微镜测定心肌细胞钙火花,after-transient,并用膜片钳测量L-型钙通道电流,同时加入兰尼定受体阻断剂兰尼定之后再计算快速刺激后产生after-contraction的细胞百分率。
结果：
BPA剂量—效应曲线：以心肌细胞收缩率为指标,针对雌性大鼠心室肌细胞,BPA的剂量—效应曲线与E2结果相似均呈倒“U”状,最大效应浓度为10-9M。
心肌细胞收缩率：BPA和E2均能使雌性大鼠心室肌细胞收缩率明显增加(p<0.01),两者共存时能使收缩率进一步增加(与对照组比较p<0.01,与BPA和E2组比较均p<0.01)。而对于雄性大鼠心室肌细胞,不论是BPA还是E2均不影响心肌细胞的收缩率。
心肌细胞钙瞬变：针对于雌性大鼠心肌细胞,BPA和E2均能明显增加钙瞬变的峰值(与正常对照组比较p<0.01)而BPA+E2组钙瞬变峰值进一步增加(与正常对照组比较p<0.01,与BPA和E2组比较均p<0.01)。而对于钙瞬变tau值,BPA和E2均使其缩短,BPA+E2使其进一步缩短。(与正常对照组比较p<0.01,与BPA和E2组比较均p<0.01)
稳态及静态心肌细胞肌浆网钙负荷：稳态下BPA能使雌性大鼠心肌细胞的咖啡因诱导钙瞬变峰值即肌浆网钙负荷由3.87±0.24增加至4.87±0.28(p<0.05)；E2组峰值也明显增加(p<0.01),BPA+E2组进一步增加至5.71±0.38(与正常对照组比较p<0.01)。而各组tau值结果无明显变化。因此利用钙瞬变与咖啡因诱导的钙瞬变峰值相比可得出每次电刺激时肌浆网内钙离子释放的比例,结果显示BPA可使这一比例上升至0.76±0.03(p<0.01)；E2与BPA组结果大致相同为0.80±0.03(与正常对照组比较p<0.01)；BPA+E2组也上升至0.82±0.03(与正常对照组比较p<0.01)。而静态时各组心肌细胞肌浆网钙负荷峰值不变。
心肌细胞钙火花：正常情况下,雌性大鼠心室肌细胞钙火花频率较低,经BPA处理后钙火花频率由3.18±0.17/100μm/sec增加至4.91±0.42/100μm/sec(p<0.01),加入E2后钙火花的频率也明显增加由2.76±0.27/100μm/sec增加至4.30±0.33/100μm/sec (p<0.01)而钙火花峰值各组之间无明显变化。
after-contraction:雌性大鼠心室肌细胞经2Hz的高频率快速刺激后,正常情况下一般不出现自发性after-contraction,经BPA处理后上升至13.79%(p<0.05), BPA+E2组进一步上升至28.13%(与正常对照组和BPA组比较均p<0.01),而加入兰尼定受体阻断剂ryanodine10-8M之后,BPA组出现after-contraction的细胞百分率回降为0, BPA+E2+ryanodine组回降到至1.92%(与正常对照组比较p>0.05)
after-transient:与after-contraction结果相似,正常雌性大鼠心室肌细胞高频率刺激后出现after-transient的细胞百分率为0,加入BPA后上升至17.02%(p<0.01),BPA浓度加倍后并不见百分率进一步升高,为15.79(与正常对照组比较p<0.01,与BPA组比较p>0.05),E2组结果与BPA相似,能明显增加出现after-transient的细胞百分率(p<0.05),E2浓度加倍后也不出现百分率进一步增加,而BPA和E2共存时after-transient的细胞百分率进一步明显增加为34.85%(与正常对照组比较p<0.01,与BPA及E2组比较均p<0.05)
心肌细胞L型钙电流：BPA对正常雌性大鼠心室肌细胞的L型钙电流的峰值无影响。
讨论与结论：
BPA广泛存在于人类的日常生活中,与各种疾病发病之间有着密切的联系,流行病学研究发现作为雌激素的内分泌干扰化合物人体尿液中的BPA含量与心血管事件之间有着一定的相关性。但其与心律失常之间是否有关联,尚未见报道。本研究发现,BPA能增加大鼠心肌细胞after-contraction和after-transient等心律失常事件延迟后除极的相关指标,即BPA可能有致心律失常作用,且这一作用具有雌性性别特异性,进一步研究发现静止状态下BPA不增加肌浆网内钙负荷,但能增加钙火花的频率,由此可见静态时BPA能通过增加兰尼定受体的开放率从而增加肌浆网钙离子漏,从而增加钙火花,但心脏不可能一直处于静止状态,因此我们检测稳态心肌细胞的肌浆网钙负荷发现,肌浆网钙负荷增加、钙瞬变峰值增加,且在有兰尼定受体阻断剂兰尼定存在时,BPA并不增加延迟后除极的发生率,可推测在BPA存在情况下,肌浆网内的钙负荷随着心脏的每次收缩逐步增加,并且同时伴随着兰尼定受体的开放率增加,从而增加心肌细胞肌浆网的钙离子漏,诱发after-contraction和after-transient等延迟后除极反应,促进心律失常发生。本研究还发现在有E2存在时,BPA能进一步加重延迟后除极,而无论是双倍浓度的E2还是BPA并不具备这一作用,由此可推测BPA与E2之间并非是一种单纯的叠加作用,而应该是一种协同作用。本部分研究主要探讨了BPA对心室肌细胞的促心律失常作用及其电生理机制。为了进一步深入了解BPA这一作用的分子机制,我们进行了下一部分的实验研究。
第二部分：低剂量BPA促雌性大鼠心肌细胞心律失常发生的分子机制
目的：
本部分研究以成年雌性大鼠心肌细胞为实验对象,检测ERα和β兴奋剂对成年雌性大鼠心肌细胞收缩率及after-transient的作用以及ERα和β的兴奋剂和拮抗剂对BPA作用的影响,并观察BPA对ERp基因敲除小鼠心室肌细胞after-transient的作用。从而试图探讨ER的两个α和β亚型在BPA的急性促心律失常作用中所扮演的角色,阐明BPA的分子作用机制。
方法：
成年大鼠心肌细胞经ERα兴奋剂PPT和ERβ兴奋剂DPN处理后利用Video edge detection system检测心肌细胞的收缩率,并利用共聚焦显微镜检测after-transient,同时ERa兴奋剂PPT、ERβ兴奋剂DPN、ERα拮抗剂MPP、ERp拮抗剂PHTPP与BPA+E2共同处理成年大鼠心肌细胞2分钟后,用共聚焦显微镜检测after-transient。用酶解法分离出ERβ基因敲除小鼠心室肌细胞,BPA+E2处理2分钟后,检测after-transient的发生率。
结果：
ERβ兴奋剂DPN对雌性大鼠心肌细胞收缩率的剂量—效应曲线：DPN的浓度分别为10-11M、10-10M、10-9M、10-8M、10-7M,结果显示DPN的剂量-效应曲线为直线型,随着剂量的逐步增加,DPN对心肌细胞收缩率的增强作用越大,效应最大时的浓度为10-7M。
ERa兴奋剂PPT对雌性大鼠心肌细胞收缩率的剂量一效应曲线：以心肌细胞收缩率为检测指标,观察PPT的剂量—效应曲线,PPT浓度分别为10-9M、10-8M、10-7M、10-6M,随着浓度的逐步上升,PPT抑制雌性大鼠心肌细胞收缩率的作用逐渐增加。最大效应浓度为10-6M。并且PPT能抑制E2增加心肌细胞收缩率。
ERa阻断剂MPP及ERβ阻断剂PHTPP对E2及BPA增强雌性大鼠心肌细胞收缩率作用的影响：BPA能增强雌性成年大鼠心肌细胞的收缩率,MPP对BPA的作用无影响,而5X10-6M的PHTPP能抑制BPA增强雌性大鼠心肌细胞收缩率的作用。同样MPP及PHTPP对E2增强雌性大鼠心肌细胞收缩率作用的影响与BPA一致。
ERα及ERβ阻断剂和兴奋剂对雄性大鼠心肌细胞收缩率的影响：对于雄性大鼠心肌细胞,E2对其收缩率无影响,同时加入E2及ERα的阻断剂(10-6M) MPP后可增加心肌细胞收缩率(p<0.01),而同时加入E2及ERβ阻断剂PHTPP则可减弱心肌细胞收缩率,ERβ兴奋剂DPN则能增加大鼠心肌细胞的收缩率(p<0.01),ERα兴奋剂PPT对大鼠心肌细胞收缩率发挥抑制作用。
BPA、ERα和ERβ兴奋剂对野生型雌性小鼠及ERβ基因敲除小鼠心肌细胞收缩率的作用：BPA及E2均能增加野生型雌性小鼠心肌细胞收缩率,(p<0.01),同样,ERβ兴奋剂DPN也能增加雌性小鼠心肌细胞收缩率(p<0.01),而ERα兴奋剂PPT则减弱雌性小鼠心肌细胞收缩率。而对于ERβ基因敲除雌性小鼠心肌细胞,BPA及E2均对心肌细胞收缩率无影响,ERβ兴奋剂DPN也对其无作用。而ERα兴奋剂PPT则能降低基因敲除小鼠心肌细胞收缩率
ERα兴奋剂和ERβ阻断剂对BPA促雌性大鼠心肌细胞产生after-transient的影响：BPA+E2能增加雌性大鼠心肌细胞after-transient的发生率(p<0.01),而ERα兴奋剂PPT和ERβ阻断剂PHTPP均能抑制BPA+E2促after-transient发生的作用,尤其是PHTPP(与BPA+E2组及BPA+E2+PPT组比较均p<0.01)
BPA及ERβ兴奋剂对雄性大鼠心肌细胞产生after-transient的影响：与雌性大鼠结果一样,BPA并不增加雄性大鼠心肌细胞after-transient的发生率(p>0.05),既使在BPA和E2同时作用时,其发生率也为0。而DPN却能兴奋雄性大鼠心肌细胞ERβ,增加after-transient发生率(与正常对照组比较p<0.05)。
BPA对野生型及ERβ基因敲除雌性小鼠心肌细胞产生after-transient的影响：BPA+E2能增加野生型雌性小鼠心肌细胞经高频率刺激后产生after-transient的概率,而对于ERβ基因敲除雌性小鼠心肌细胞来说,BPA+E2并不增加其after-transient的发生概率。
讨论与结论：
心肌细胞膜上存在着雌激素受体两个亚型ERα和β,雌激素结合这两个受体后可通过激活细胞内的一系列信号通路发挥作用,也可通过激活细胞内的信号通路后作用于核受体从而诱导基因表达,从而发挥作用。前一作用发生得比较快,即刻就可起效,而后一作用须经过一段时间后才能表现出来。BPA作为雌激素的内分泌干扰化合物也能与这两种受体结合。本研究通过加入ERα和β的兴奋剂和拮抗剂,同时结合ERβ基因敲除小鼠模型,从正反两方面阐述这两种受体如何介导BPA的促心律失常作用。研究结果发现,ERα兴奋剂能抑制雌性成年大鼠心肌细胞收缩率,抑制BPA+E2的促细胞发生延迟后除极作用,同时ERα拮抗剂则能增加雌性大鼠心肌细胞收缩率。增加心肌细胞延迟后除极发生概率。与之相反,ERβ兴奋剂则能增强雌性大鼠心肌细胞收缩率,促进心肌细胞发生延迟后除极,而其拮抗剂则能减弱雌性大鼠心肌细胞收缩率,抑制BPA+E2的促延迟后除极作用。而对于雄性大鼠心肌细胞,激活ERα能抑制心肌细胞收缩率,而激活ERβ则能增加收缩率,有趣的是激活ERβ还能增加心肌细胞after-transient这一延迟后除极的发生概率。ERβ基因敲除小鼠结果显示,BPA+E2并不增加其心肌细胞收缩率和延迟后除极的发生概率,ERβ兴奋剂也对其无作用。以上结果可以说明,BPA主要通过作用于ERβ而发挥促心律失常作用,相反,作用于ERα后能抑制其促心律失常作用。前两部分均以单个心肌细胞为研究对象,但整体心脏的生理特征与单个细胞之间还是存在着一定的差异性,因此下一部分,我们以离体的整体心脏作为研究对象来观察BPA对心律失常的影响。
第三部分：急性状态下低剂量BPA对体外整体正常心脏电生理及心脏缺氧／再灌注损伤心律失常的影响
目的：
本部分研究以体外整体心脏为研究对象,观察正常情况以及在有异丙肾上腺素(Isoproterenol,ISO)存在下,BPA+E2对心电图的影响,同时以缺氧／再灌注损伤心脏为模型,观察BPA对缺氧／再灌注损伤性室性心律失常的影响。从整体心脏水平阐述BPA的促心律失常作用。
方法：
按实验要求分为正常对照组,BPA+E2组,ISO组及BPA+E2+ISO组,雌性和雄性成年大鼠处死后取出心脏,悬挂于Langendorff灌流系统上进行灌注,待心脏状态稳定后,开始记录各组体外整体心脏的心电图。建立缺氧／再灌注模型,雌性成年大鼠心脏悬挂于Langendorff系统上灌注至稳定状态后,停止灌注,全心脏缺氧20分钟后,用正常溶液或BPA+E2组溶液进行再灌注,同时记录心电图,再灌一小时后,将心脏取下,TTC染色,计算梗死面积。
结果：
BPA+E2对雌性大鼠心电图的影响：正常离体灌注心脏不发生任何形式的室性心律失常。加入BPA+E2后室性心律失常的发生率也很低(0 vs 1.22±0.72个/20min p>0.05),ISO组也偶可见室性心律失常的发生,但与正常组比较统计学上无差异(1.60±1.10个/20min vs 0,p>0.05).有ISO存在时,BPA+E2能明显增加室性早搏的频率至6.72±2.59个/20min(与正常对照组,BPA+E2组及ISO组比较均p<0.05)
BPA+E2对雄性大鼠心电图的影响与雌性大鼠离体心脏一致,正常情况下,心脏不发生任何形式的室性心律失常,室性早搏发生的频率为0个/20min,,BPA+E2也不能使雄性大鼠体外整体心脏产生室性早搏(0个/20min),加入ISO后雄性大鼠心脏产生室性早搏的频率明显增加(6.42±2.03 vs 0个/20min,p<0.05)。而在ISO存在的基础上加入BPA+E2并不能使室性早搏发生频率增加,仅为5.71±1.92个/20min(与正常对照组比较p<0.05,与ISO组比较p>0.05)
BPA+E2对缺氧／再灌注损伤心律失常的影响缺氧20分钟再灌注后,对照组5分钟时恶性室性心律失常已停止,10分钟时基本恢复正常。而BPA+E2组,2分钟时依旧持续发生室颤,5分钟时转换为室性心动过速,直至10分钟,心律失常才消失。正常情况下,缺氧／再灌注后室性心律失常持续时间为280.10±76.62sec。加入BPA+E2后室性心律失常持续的时间延长为529.70±89.91sec(与对照组比较,p<0.05),且明显减少室性心律失常中室速的百分比(69.51±8.06% vs 90.02±5.03%,p<0.05),增加室颤的百分比(30.49±8.06% vs 9.98±5.02%,p<0.05),由此可见BPA+E2不但延长缺氧／再灌注损伤室性心律失常的持续时间,而且进一步恶化心律失常性质。
BPA+E2对缺氧／再灌注损伤心肌梗死面积的影响缺血20分钟再灌注1小时后,BPA+E2能明显缩小心肌梗死面积(p<0.05)。
讨论与结论：
前两部分阐述了BPA促心肌细胞发生延迟后除极的作用及其电生理和分子机制。心律失常的发生机制有三类,即自律性增强、折返和触发活动,触发活动又包含有早期后除极和延迟后除极。心室肌细胞延迟后除极发生概率增加,在整体心脏就表现为心电图上的室性心律失常即室性早搏,室性心动过速及室颤,本部分检测了BPA对离体整体心脏心电图的影响,结果显示正常情况下,给予BPA+E2后心电图未见明显室性心律失常发生,而在ISO模拟的急性应激状态下,BPA+E2能明显增加雌性大鼠室性早搏发生频率,而对于雄性大鼠心脏,即使在急性应激状态下,也不会增加室性早搏发生频率。因此本研究以临床上常见的应激状态即缺氧／再灌注损伤为模型,观察病理状态下BPA对整体心脏心律失常的影响,结果显示BPA+E2能明显延长缺氧／再灌注后室性心律失常的持续时间,增加心律失常中室颤发生百分比,进一步恶化室性心律失常。对心梗面积进行统计发现BPA+E2能明显缩小再灌注损伤心肌梗死面积,这实验结果与其它已报道的实验结果相一致。
结合这三部分实验结果,可得出以下结论。雌激素内分泌干扰化合物BPA能促使室性心律失常事件发生,且与雌二醇之间具有协同作用。其具体作用机制为BPA能结合细胞膜上的ERβ,激活一系列信号通道,增加兰尼定受体的开放及肌浆网内钙负荷,增加心肌细胞钙火花的频率,从而诱导延迟后除极活动,促进心脏室性心律失常的发生,特别是在病理状态下,这一作用尤为明显。且这一作用为雌性特异性。BPA结合细胞膜上的ERα则能抑制其促心律失常的作用。
Background
Bishphenol 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 mechanisms
Objectives:
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.+E2
The 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 Discussions
Previous 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.
1891年Aleksandr Dianin第一次人工合成了化合物双酚A (Bisphenol A,BPA)。从此揭开了人类塑料工业的新篇章。BPA是聚碳酸酯塑料及树脂的主要成分之一,五十年代以来,随着塑料业的快速发展,BPA越来越广泛地应用于人类日常生活用品中,例如塑料婴儿奶瓶,饮料瓶,牙齿封闭剂及塑料食物盒等,且人类对BPA的需求越来越高,其对人类环境的污染也日益严重,从而使得越来越多的人群接触BPA,大规模的人群调查研究发现95%以上的人群尿液中能检测出BPA,其平均浓度为10-9M左右。因此由于BPA的广泛污染而给人类带来的影响也成为各个领域的研究者高度关注的话题。目前已证实BPA能.模拟／拮抗雌激素的作用,而被认为是一种雌激素的内分泌干扰化合物。作为雌激素的内分泌干扰化合物,BPA能结合雌激素受体(Estrogen Receptor, ER)α和β。发挥由ER介导的急性反应。有研究发现BPA具有促肿瘤,糖尿病和肥胖等发生发展作用,对机体的内分泌,生殖系统也能产生影响,而且认为人体尿液中的BPA浓度与心血管事件之间有着一定的相关性。
根据目前的研究现状与结果,我们试图证实低剂量BPA在急性状态下对大鼠单个心肌细胞及离体心脏可能具有促心律失常作用。且由于BPA为性激素内分泌干扰物,因此其对雌雄性大鼠可能发挥着不同的作用。这一作用可能是通过结合于细胞膜上的ER,快速激活相关的信号通道而实现的。此外,本研究还试图探讨在心脏缺氧／再灌注损伤时,BPA对灌注后心律失常的影响。
为了证实这一推测。本研究方案如下：
首先,以体外分离的成年大鼠心室肌细胞为研究对象,以心肌细胞收缩率为指标,观察BPA对不同性别大鼠心肌细胞的作用有何不同,同时在急性状态下单纯给予BPA或在有雌二醇(Estrodial, E2)存在时给予BPA,用离子成像系统和共聚焦显微镜等手段检测心肌细胞钙火花,钙瞬变,肌浆网钙负荷以及延迟后除极相关指标after-contraction和after-transient, L型钙电流等钙动力学相关指标的变化,并观察兰尼定受体阻断剂兰尼定是否影响BPA的作用,初步探讨BPA对单个心肌细胞的促心律失常作用及其是否具有性别特异性,并探讨其发生的电生理作用机制。
其次,以ERβ基因敲除小鼠为实验模型,观察BPA对心肌细胞快速刺激后产生after-transient的影响。同时检测ERα和β兴奋剂对成年雌性大鼠心肌细胞收缩率及after-transient的作用,并在给予BPA的同时给予ERα和β拮抗剂,观察二者是否影响BPA的作用,从而试图解释ER的两个亚型α和β如何介导在BPA的急性促心律失常作用,初步探讨BPA的分子作用机制。
最后,以体外灌注的整体心脏作为研究对象,检测BPA对其心电图的作用,同时观察缺氧／再灌注损伤时,BPA对缺氧／再灌注损伤室性心律失常及梗死面积的影响,阐述BPA对离体整体心脏电生理的影响。从而以单个细胞结合整体心脏,正常生理状态结合病理状态,全面地探讨低剂量BPA对雌性大鼠心脏的促心律失常作用。
第一部分：低剂量BPA对雌性大鼠心肌细胞的急性促心律失常作用及其电生理机制
目的：
以体外分离的成年大鼠心肌细胞为研究对象,通过离子成像系统,共聚焦显微镜及膜片钳等检测手段观察心肌细胞收缩率,心肌细胞钙火花,钙瞬变,肌浆网钙负荷以及延迟后除极指标after-contraction和after-transient, L型钙电流等钙动力学相关指标的变化,初步探讨BPA促进单个心肌细胞心律失常发生的电生理机制及其性别特异性。
方法：
雌雄性成年大鼠以酶解法分离出单个的心室肌细胞,再按实验要求分为四组,正常对照组,BPA组,E2组,BPA+E2组,各组细胞分别经药物处理2分钟后,用Video edge detection system测量心肌细胞收缩率及after-contraction,用离子成像系统测定心肌细胞钙瞬变,肌浆网钙负荷,用共聚焦显微镜测定心肌细胞钙火花,after-transient,并用膜片钳测量L-型钙通道电流,同时加入兰尼定受体阻断剂兰尼定之后再计算快速刺激后产生after-contraction的细胞百分率。
结果：
BPA剂量—效应曲线：以心肌细胞收缩率为指标,针对雌性大鼠心室肌细胞,BPA的剂量—效应曲线与E2结果相似均呈倒“U”状,最大效应浓度为10-9M。
心肌细胞收缩率：BPA和E2均能使雌性大鼠心室肌细胞收缩率明显增加(p<0.01),两者共存时能使收缩率进一步增加(与对照组比较p<0.01,与BPA和E2组比较均p<0.01)。而对于雄性大鼠心室肌细胞,不论是BPA还是E2均不影响心肌细胞的收缩率。
心肌细胞钙瞬变：针对于雌性大鼠心肌细胞,BPA和E2均能明显增加钙瞬变的峰值(与正常对照组比较p<0.01)而BPA+E2组钙瞬变峰值进一步增加(与正常对照组比较p<0.01,与BPA和E2组比较均p<0.01)。而对于钙瞬变tau值,BPA和E2均使其缩短,BPA+E2使其进一步缩短。(与正常对照组比较p<0.01,与BPA和E2组比较均p<0.01)
稳态及静态心肌细胞肌浆网钙负荷：稳态下BPA能使雌性大鼠心肌细胞的咖啡因诱导钙瞬变峰值即肌浆网钙负荷由3.87±0.24增加至4.87±0.28(p<0.05)；E2组峰值也明显增加(p<0.01),BPA+E2组进一步增加至5.71±0.38(与正常对照组比较p<0.01)。而各组tau值结果无明显变化。因此利用钙瞬变与咖啡因诱导的钙瞬变峰值相比可得出每次电刺激时肌浆网内钙离子释放的比例,结果显示BPA可使这一比例上升至0.76±0.03(p<0.01)；E2与BPA组结果大致相同为0.80±0.03(与正常对照组比较p<0.01)；BPA+E2组也上升至0.82±0.03(与正常对照组比较p<0.01)。而静态时各组心肌细胞肌浆网钙负荷峰值不变。
心肌细胞钙火花：正常情况下,雌性大鼠心室肌细胞钙火花频率较低,经BPA处理后钙火花频率由3.18±0.17/100μm/sec增加至4.91±0.42/100μm/sec(p<0.01),加入E2后钙火花的频率也明显增加由2.76±0.27/100μm/sec增加至4.30±0.33/100μm/sec (p<0.01)而钙火花峰值各组之间无明显变化。
after-contraction:雌性大鼠心室肌细胞经2Hz的高频率快速刺激后,正常情况下一般不出现自发性after-contraction,经BPA处理后上升至13.79%(p<0.05), BPA+E2组进一步上升至28.13%(与正常对照组和BPA组比较均p<0.01),而加入兰尼定受体阻断剂ryanodine10-8M之后,BPA组出现after-contraction的细胞百分率回降为0, BPA+E2+ryanodine组回降到至1.92%(与正常对照组比较p>0.05)
after-transient:与after-contraction结果相似,正常雌性大鼠心室肌细胞高频率刺激后出现after-transient的细胞百分率为0,加入BPA后上升至17.02%(p<0.01),BPA浓度加倍后并不见百分率进一步升高,为15.79(与正常对照组比较p<0.01,与BPA组比较p>0.05),E2组结果与BPA相似,能明显增加出现after-transient的细胞百分率(p<0.05),E2浓度加倍后也不出现百分率进一步增加,而BPA和E2共存时after-transient的细胞百分率进一步明显增加为34.85%(与正常对照组比较p<0.01,与BPA及E2组比较均p<0.05)
心肌细胞L型钙电流：BPA对正常雌性大鼠心室肌细胞的L型钙电流的峰值无影响。
讨论与结论：
BPA广泛存在于人类的日常生活中,与各种疾病发病之间有着密切的联系,流行病学研究发现作为雌激素的内分泌干扰化合物人体尿液中的BPA含量与心血管事件之间有着一定的相关性。但其与心律失常之间是否有关联,尚未见报道。本研究发现,BPA能增加大鼠心肌细胞after-contraction和after-transient等心律失常事件延迟后除极的相关指标,即BPA可能有致心律失常作用,且这一作用具有雌性性别特异性,进一步研究发现静止状态下BPA不增加肌浆网内钙负荷,但能增加钙火花的频率,由此可见静态时BPA能通过增加兰尼定受体的开放率从而增加肌浆网钙离子漏,从而增加钙火花,但心脏不可能一直处于静止状态,因此我们检测稳态心肌细胞的肌浆网钙负荷发现,肌浆网钙负荷增加、钙瞬变峰值增加,且在有兰尼定受体阻断剂兰尼定存在时,BPA并不增加延迟后除极的发生率,可推测在BPA存在情况下,肌浆网内的钙负荷随着心脏的每次收缩逐步增加,并且同时伴随着兰尼定受体的开放率增加,从而增加心肌细胞肌浆网的钙离子漏,诱发after-contraction和after-transient等延迟后除极反应,促进心律失常发生。本研究还发现在有E2存在时,BPA能进一步加重延迟后除极,而无论是双倍浓度的E2还是BPA并不具备这一作用,由此可推测BPA与E2之间并非是一种单纯的叠加作用,而应该是一种协同作用。本部分研究主要探讨了BPA对心室肌细胞的促心律失常作用及其电生理机制。为了进一步深入了解BPA这一作用的分子机制,我们进行了下一部分的实验研究。
第二部分：低剂量BPA促雌性大鼠心肌细胞心律失常发生的分子机制
目的：
本部分研究以成年雌性大鼠心肌细胞为实验对象,检测ERα和β兴奋剂对成年雌性大鼠心肌细胞收缩率及after-transient的作用以及ERα和β的兴奋剂和拮抗剂对BPA作用的影响,并观察BPA对ERp基因敲除小鼠心室肌细胞after-transient的作用。从而试图探讨ER的两个α和β亚型在BPA的急性促心律失常作用中所扮演的角色,阐明BPA的分子作用机制。
方法：
成年大鼠心肌细胞经ERα兴奋剂PPT和ERβ兴奋剂DPN处理后利用Video edge detection system检测心肌细胞的收缩率,并利用共聚焦显微镜检测after-transient,同时ERa兴奋剂PPT、ERβ兴奋剂DPN、ERα拮抗剂MPP、ERp拮抗剂PHTPP与BPA+E2共同处理成年大鼠心肌细胞2分钟后,用共聚焦显微镜检测after-transient。用酶解法分离出ERβ基因敲除小鼠心室肌细胞,BPA+E2处理2分钟后,检测after-transient的发生率。
结果：
ERβ兴奋剂DPN对雌性大鼠心肌细胞收缩率的剂量—效应曲线：DPN的浓度分别为10-11M、10-10M、10-9M、10-8M、10-7M,结果显示DPN的剂量-效应曲线为直线型,随着剂量的逐步增加,DPN对心肌细胞收缩率的增强作用越大,效应最大时的浓度为10-7M。
ERa兴奋剂PPT对雌性大鼠心肌细胞收缩率的剂量一效应曲线：以心肌细胞收缩率为检测指标,观察PPT的剂量—效应曲线,PPT浓度分别为10-9M、10-8M、10-7M、10-6M,随着浓度的逐步上升,PPT抑制雌性大鼠心肌细胞收缩率的作用逐渐增加。最大效应浓度为10-6M。并且PPT能抑制E2增加心肌细胞收缩率。
ERa阻断剂MPP及ERβ阻断剂PHTPP对E2及BPA增强雌性大鼠心肌细胞收缩率作用的影响：BPA能增强雌性成年大鼠心肌细胞的收缩率,MPP对BPA的作用无影响,而5X10-6M的PHTPP能抑制BPA增强雌性大鼠心肌细胞收缩率的作用。同样MPP及PHTPP对E2增强雌性大鼠心肌细胞收缩率作用的影响与BPA一致。
ERα及ERβ阻断剂和兴奋剂对雄性大鼠心肌细胞收缩率的影响：对于雄性大鼠心肌细胞,E2对其收缩率无影响,同时加入E2及ERα的阻断剂(10-6M) MPP后可增加心肌细胞收缩率(p<0.01),而同时加入E2及ERβ阻断剂PHTPP则可减弱心肌细胞收缩率,ERβ兴奋剂DPN则能增加大鼠心肌细胞的收缩率(p<0.01),ERα兴奋剂PPT对大鼠心肌细胞收缩率发挥抑制作用。
BPA、ERα和ERβ兴奋剂对野生型雌性小鼠及ERβ基因敲除小鼠心肌细胞收缩率的作用：BPA及E2均能增加野生型雌性小鼠心肌细胞收缩率,(p<0.01),同样,ERβ兴奋剂DPN也能增加雌性小鼠心肌细胞收缩率(p<0.01),而ERα兴奋剂PPT则减弱雌性小鼠心肌细胞收缩率。而对于ERβ基因敲除雌性小鼠心肌细胞,BPA及E2均对心肌细胞收缩率无影响,ERβ兴奋剂DPN也对其无作用。而ERα兴奋剂PPT则能降低基因敲除小鼠心肌细胞收缩率
ERα兴奋剂和ERβ阻断剂对BPA促雌性大鼠心肌细胞产生after-transient的影响：BPA+E2能增加雌性大鼠心肌细胞after-transient的发生率(p<0.01),而ERα兴奋剂PPT和ERβ阻断剂PHTPP均能抑制BPA+E2促after-transient发生的作用,尤其是PHTPP(与BPA+E2组及BPA+E2+PPT组比较均p<0.01)
BPA及ERβ兴奋剂对雄性大鼠心肌细胞产生after-transient的影响：与雌性大鼠结果一样,BPA并不增加雄性大鼠心肌细胞after-transient的发生率(p>0.05),既使在BPA和E2同时作用时,其发生率也为0。而DPN却能兴奋雄性大鼠心肌细胞ERβ,增加after-transient发生率(与正常对照组比较p<0.05)。
BPA对野生型及ERβ基因敲除雌性小鼠心肌细胞产生after-transient的影响：BPA+E2能增加野生型雌性小鼠心肌细胞经高频率刺激后产生after-transient的概率,而对于ERβ基因敲除雌性小鼠心肌细胞来说,BPA+E2并不增加其after-transient的发生概率。
讨论与结论：
心肌细胞膜上存在着雌激素受体两个亚型ERα和β,雌激素结合这两个受体后可通过激活细胞内的一系列信号通路发挥作用,也可通过激活细胞内的信号通路后作用于核受体从而诱导基因表达,从而发挥作用。前一作用发生得比较快,即刻就可起效,而后一作用须经过一段时间后才能表现出来。BPA作为雌激素的内分泌干扰化合物也能与这两种受体结合。本研究通过加入ERα和β的兴奋剂和拮抗剂,同时结合ERβ基因敲除小鼠模型,从正反两方面阐述这两种受体如何介导BPA的促心律失常作用。研究结果发现,ERα兴奋剂能抑制雌性成年大鼠心肌细胞收缩率,抑制BPA+E2的促细胞发生延迟后除极作用,同时ERα拮抗剂则能增加雌性大鼠心肌细胞收缩率。增加心肌细胞延迟后除极发生概率。与之相反,ERβ兴奋剂则能增强雌性大鼠心肌细胞收缩率,促进心肌细胞发生延迟后除极,而其拮抗剂则能减弱雌性大鼠心肌细胞收缩率,抑制BPA+E2的促延迟后除极作用。而对于雄性大鼠心肌细胞,激活ERα能抑制心肌细胞收缩率,而激活ERβ则能增加收缩率,有趣的是激活ERβ还能增加心肌细胞after-transient这一延迟后除极的发生概率。ERβ基因敲除小鼠结果显示,BPA+E2并不增加其心肌细胞收缩率和延迟后除极的发生概率,ERβ兴奋剂也对其无作用。以上结果可以说明,BPA主要通过作用于ERβ而发挥促心律失常作用,相反,作用于ERα后能抑制其促心律失常作用。前两部分均以单个心肌细胞为研究对象,但整体心脏的生理特征与单个细胞之间还是存在着一定的差异性,因此下一部分,我们以离体的整体心脏作为研究对象来观察BPA对心律失常的影响。
第三部分：急性状态下低剂量BPA对体外整体正常心脏电生理及心脏缺氧／再灌注损伤心律失常的影响
目的：
本部分研究以体外整体心脏为研究对象,观察正常情况以及在有异丙肾上腺素(Isoproterenol,ISO)存在下,BPA+E2对心电图的影响,同时以缺氧／再灌注损伤心脏为模型,观察BPA对缺氧／再灌注损伤性室性心律失常的影响。从整体心脏水平阐述BPA的促心律失常作用。
方法：
按实验要求分为正常对照组,BPA+E2组,ISO组及BPA+E2+ISO组,雌性和雄性成年大鼠处死后取出心脏,悬挂于Langendorff灌流系统上进行灌注,待心脏状态稳定后,开始记录各组体外整体心脏的心电图。建立缺氧／再灌注模型,雌性成年大鼠心脏悬挂于Langendorff系统上灌注至稳定状态后,停止灌注,全心脏缺氧20分钟后,用正常溶液或BPA+E2组溶液进行再灌注,同时记录心电图,再灌一小时后,将心脏取下,TTC染色,计算梗死面积。
结果：
BPA+E2对雌性大鼠心电图的影响：正常离体灌注心脏不发生任何形式的室性心律失常。加入BPA+E2后室性心律失常的发生率也很低(0 vs 1.22±0.72个/20min p>0.05),ISO组也偶可见室性心律失常的发生,但与正常组比较统计学上无差异(1.60±1.10个/20min vs 0,p>0.05).有ISO存在时,BPA+E2能明显增加室性早搏的频率至6.72±2.59个/20min(与正常对照组,BPA+E2组及ISO组比较均p<0.05)
BPA+E2对雄性大鼠心电图的影响与雌性大鼠离体心脏一致,正常情况下,心脏不发生任何形式的室性心律失常,室性早搏发生的频率为0个/20min,,BPA+E2也不能使雄性大鼠体外整体心脏产生室性早搏(0个/20min),加入ISO后雄性大鼠心脏产生室性早搏的频率明显增加(6.42±2.03 vs 0个/20min,p<0.05)。而在ISO存在的基础上加入BPA+E2并不能使室性早搏发生频率增加,仅为5.71±1.92个/20min(与正常对照组比较p<0.05,与ISO组比较p>0.05)
BPA+E2对缺氧／再灌注损伤心律失常的影响缺氧20分钟再灌注后,对照组5分钟时恶性室性心律失常已停止,10分钟时基本恢复正常。而BPA+E2组,2分钟时依旧持续发生室颤,5分钟时转换为室性心动过速,直至10分钟,心律失常才消失。正常情况下,缺氧／再灌注后室性心律失常持续时间为280.10±76.62sec。加入BPA+E2后室性心律失常持续的时间延长为529.70±89.91sec(与对照组比较,p<0.05),且明显减少室性心律失常中室速的百分比(69.51±8.06% vs 90.02±5.03%,p<0.05),增加室颤的百分比(30.49±8.06% vs 9.98±5.02%,p<0.05),由此可见BPA+E2不但延长缺氧／再灌注损伤室性心律失常的持续时间,而且进一步恶化心律失常性质。
BPA+E2对缺氧／再灌注损伤心肌梗死面积的影响缺血20分钟再灌注1小时后,BPA+E2能明显缩小心肌梗死面积(p<0.05)。
讨论与结论：
前两部分阐述了BPA促心肌细胞发生延迟后除极的作用及其电生理和分子机制。心律失常的发生机制有三类,即自律性增强、折返和触发活动,触发活动又包含有早期后除极和延迟后除极。心室肌细胞延迟后除极发生概率增加,在整体心脏就表现为心电图上的室性心律失常即室性早搏,室性心动过速及室颤,本部分检测了BPA对离体整体心脏心电图的影响,结果显示正常情况下,给予BPA+E2后心电图未见明显室性心律失常发生,而在ISO模拟的急性应激状态下,BPA+E2能明显增加雌性大鼠室性早搏发生频率,而对于雄性大鼠心脏,即使在急性应激状态下,也不会增加室性早搏发生频率。因此本研究以临床上常见的应激状态即缺氧／再灌注损伤为模型,观察病理状态下BPA对整体心脏心律失常的影响,结果显示BPA+E2能明显延长缺氧／再灌注后室性心律失常的持续时间,增加心律失常中室颤发生百分比,进一步恶化室性心律失常。对心梗面积进行统计发现BPA+E2能明显缩小再灌注损伤心肌梗死面积,这实验结果与其它已报道的实验结果相一致。
结合这三部分实验结果,可得出以下结论。雌激素内分泌干扰化合物BPA能促使室性心律失常事件发生,且与雌二醇之间具有协同作用。其具体作用机制为BPA能结合细胞膜上的ERβ,激活一系列信号通道,增加兰尼定受体的开放及肌浆网内钙负荷,增加心肌细胞钙火花的频率,从而诱导延迟后除极活动,促进心脏室性心律失常的发生,特别是在病理状态下,这一作用尤为明显。且这一作用为雌性特异性。BPA结合细胞膜上的ERα则能抑制其促心律失常的作用。
Background
Bishphenol 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 mechanisms
Objectives:
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.+E2
The 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 Discussions
Previous 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.
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