不同浓度的17β-雌二醇对心肌细胞H9c2细胞内游离离子稳态的影响及机制研究
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摘要
自上世纪以来的流行病学研究表明,心血管疾病的发病率和死亡率存在着性别差异。绝经前女性心血管疾病的发病率明显低于同龄男性。而且,不管是由于自然的因素还是外科手术抑或是卵巢功能受损从而导致雌激素分泌的停滞,都会使女性患心血管疾病的危险性增加。女性心脏疾病的发病比男性平均晚10年,绝经后妇女心血管疾病的发病率是同龄的绝经前妇女的4倍以上。雌激素水平下降是绝经后妇女最主要的生理变化。实验研究证实,天然雌激素如17β-雌二醇可直接作用于心脏或影响血管系统从而对心血管疾病的发生产生影响。近年来研究认为雌激素可通过防止动脉硬化形成和血管重塑而起保护作用,包括雌激素的抗凋亡、抗氧化,改变脂蛋白代谢、血小板的粘附和聚集,血管内皮激活一氧化氮合成从而导致血管舒张,抑制心肌肥大和减少心肌缺血等。因此,男性与绝经前同龄女性之间心血管疾病的发病率和死亡率之间的差异主要是由于雌激素的保护作用。然而,绝经后女性大样本随机临床试验的研究表明雌激素和孕激素的联合使用结果对激素替代的心血管保护作用提出了质疑,认为激素替代不仅不对心血管有保护作用,反而增加心血管疾病的发病率和死亡率,引起心脏疾病、乳腺癌、脑血栓和痴呆等发病的危险性。这些有关雌激素对心血管作用负面研究结果引发了人们对激素替代治疗的质疑。为更好地解决激素替代治疗引发的公共卫生危机,关键是弄清楚雌激素对心血管系统的快效应和慢效应的复杂机制,因此需要对雌激素的正效应和负作用进行全面评估。雌激素对血管系统的作用已有较全面的认识,但对心脏的直接作用还知之甚少。近年来,研究的焦点已开始从血管系统转向心肌细胞。一般认为,细胞内离子稳态对维持心肌细胞的正常功能起着重要的作用,心脏正常的收缩功能有赖于心肌细胞离子稳态和激活-收缩耦联。雌激素对心肌细胞离子通道的瞬时效应已有广泛的研究,然而对其长期效应却知之甚少,因此阐明雌激素影响心肌细胞内离子稳态作用机制是十分必需的。
在我们以往的研究中,利用cDNA微阵列技术观察到：去势3个月后,左室心肌细胞的上百个基因的表达发生改变,其中包括Na+,K+-ATP酶、Ca2+-ATP酶、碳酸酐酶、L型钙通道蛋白、延迟整流钾通道蛋白和电压门控钠通道蛋白以及Na+-H+交换蛋白和Na+-Ca2+交换蛋白等的表达。心肌细胞电解质稳定对其功能发挥起至关重要的作用。对心肌细胞H9c2的研究发现生理浓度的雌激素能上调Na+,K+-ATP酶、肌浆网Ca2+-ATP酶和碳酸酐酶的表达和活性,这些酶均可能参与细胞内外电解质平衡的调节。上述的实验结果提示,生理浓度的雌激素水平可能维护心肌细胞内的离子浓度、pH值和膜电生理等特性而保护心肌细胞；否则,会导致电解质紊乱、电生理异常、心肌细胞损伤乃至死亡。因此,本研究的目的是寻找雌激素对心肌细胞内游离离子稳态影响的直接证据。我们用鼠H9c2心肌细胞为研究对象,检测不同浓度雌激素对细胞内Ca2+、Na+、K+、H+和Cl-等离子浓度和ⅠNa、Ⅰca,L、ⅠK等离子电流的影响。
目的研究不同浓度的17β-雌二醇对心肌细胞H9c2内Ca2+、Na+、K+、H+、C1-等游离离子浓度的影响。
方法H9c2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,接着分别用荧光探针Fluo-3,SBFI, PBFI,SNARF-1,MQAE孵育细胞,用荧光法测定细胞内游离离子(Ca2+、Na+、K+、H+、C1-)浓度。
结果心肌细胞H9c2分别在O、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钙离子浓度分别是(157.5±5.7)nM,(153.8±6.2)nM,(148.5±8.7)nM,(144.5±7.8)nM,(167.5±9.0)nM。实验结果显示：与无17β-雌二醇组相比,0.01nM、1 nM和100 nM17β-雌二醇组的细胞内[Ca2+]下降(p<0.05)。无E2组和0.01 nM E2组细胞内[Ca2+]显著高于1 nM E2组(p<0.05)。1 nm和100 nM17p-雌二醇组之间细胞内[Ca2+]无显著性差别(p>0.05),而1000 nM17p-雌二醇组细胞内[Ca2+]却比无E2组和0.01 nME2组,1 nM E2组和100nM E2组有显著性升高(p<0.05)。
心肌细胞H9e2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钠离子浓度分别是(8.9±0.5)mM,(8.5±0.8)mM,(7.8±0.8)mM,(7.7±0.9)mM,(9.9±0.7)mM。0.01 nM17β-雌二醇组细胞内[Na+]与无17p-雌二醇组无显著性差异(p>0.05)。当17p-雌二醇浓度为1 nM、100 nM时,H9c2细胞内[Na+]比无17β-雌二醇时存在显著降低(p<0.05)。无17p-雌二醇组和0.01 nM17β-雌二醇组细胞内[Na+]比1 nM 17p-雌二醇组有显著性升高(p<0.05)。但是1 nM 17β-雌二醇组与100 nnM17β-雌二醇组之间[Na+]无显著性差异(p>0.05)。1000 nM17β-雌二醇组细胞内[Na+]显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钾离子浓度分别是(134.4±8.3)mM,(137.2±7.5)mM,(140.0±7.5)mM,(142.0±±6.8)mM,(133.7±9.6)mM。对H9c2细胞内的[K+],0.01 nM E2组和无E2组之间无显著性差异(p>0.05)。1 nM和100 nM 2组细胞内[K+]显著性高于无E2组(p<0.05)。1 nM E2和100 nM E2组之间亦无显著性差异(p>0.05)。但是当H9c2细胞分别在O、0.01 nM、1000 nM E2培养24h时,细胞内[K+]比1nM E2组有显著性的下降(p<0.05)。
心肌细胞H9c2分别在0、0.01nM、1nM、100nM、1000nM17p-雌二醇中培养24小时后,心肌细胞内pH分别是(7.0±0.2),(7.3±0.3),(7.44-0.2),(7.44±0.2),(6.44±0.3)。0.01 nM、1 nM、100 nM E2组的H9c2细胞内的pH显著性的高于无E2组(p<0.05)。但是在0.01 nM、1 nM、100 nM E2组之间细胞内pH值无显著性差异(p>0.05)。另外,1000nM E2组的细胞内pH显著性低于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离氯离子浓度分别是(32.3±3.2)mM,(30.6±4.8)mM,(27.6±4.5)mM,(28.5±4.4)mM,(35.7±4.0)mM。H9c2细胞内[C1-]在0.01nME2组和无E2组之间无显著性差异(p>0.05)。1 nM E2组和100 nM E2组的[Cl-]i显著性的低于无E2组(p<0.05)而1 nM E2组和100 nM E2之间H9c2细胞内[C1-]无显著性差异(p>0.05)。但是,1000 nM E2组[C1-]分别比无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组显著上升(p<0.05)。
结论研究表明1 nM和100 nM的17p-雌二醇能维持H9c2细胞内Ca2+、Na+、K+、H+、C1-等离子的浓度在生理浓度范围,1 nM和100 nM17p-雌二醇组间细胞内离子浓度无显著性差异。H9c2细胞在无E2、0.01 nME2或1000 nM E2条件下,细胞内游离离子平衡出现紊乱。因此,17p-雌二醇对H9c2细胞内Ca2+、Na+、K+、H+、C1-等游离离子浓度的稳定起着至关重要的调节作用。
目的研究不同浓度的17p-雌二醇对H9c2心肌细胞的电压门控钠电流(INa)、L型钙电流(ICa,L)、延迟整流钾电流(Ik)等离子通道电流的影响。
方法H9c2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,用全细胞膜片钳技术测定H9c2细胞的电压门控钠电流(INa)、L型钙电流(Ica,L)、延迟整流钾电流(Ik)等离子通道电流。
结果电压依赖的INa的测定：维持电位(holding potential)为-80mV,每隔10mV,连续除极至20mV。内向IN。大约在-70mV出现,在-30mV达到最大,而在+20mV逆转。用0、0.01nM、1nM、100 nM、1000nME2处理H9c2细胞24h后,其平均最大INa分别是(3.61±0.42)(pA／pF),(2.80±0.29)(pA/pF),(2.27±0.30)(pA／pF),(2.08±0.31)(pA／pF),(1.51±0.20)(pA／pF)。与无E2组相比,0.01nM、1nM、100nM、1000 nME2处理组的平均最大INa有显著性下降(p<0.05),17β-雌二醇能以浓度依赖的方式抑制INa。另外,17β-雌二醇不改变I-V曲线(n=12-16)。
17β-雌二醇对L型钙电流(Ica,L)的影响维持电位-40mV,每隔10mV连续除极至+50mV,时间180ms。ICa,L在0mV达最大。用O、0.01 nM、1nM、100nM和1000 nME2处理H9c2细胞24h后,其平均最大ICa.L分别是(9.54±0.75)(pA／pF),(7.70±0.81)(pA／pF),(6.28±0.62)(pA／pF),(5.01±0.38)(pA／pF),(4.45±0.57)(pA／pF)。与无E2组相比,0.01 nM、1nM、100 nM、1000 nM E2处理组的平均最大Ica,L有显著性下降(p<0.05)。同样的,17p-雌二醇能以浓度信赖的方式抑制Ica,L。与INa相类似,17p-雌二醇亦不影响I-V关系(n=12-16)。
17β-雌二醇对延迟整流钾电流的影响延迟整流钾电流IK测定：维持电位-40mV,每隔10mV,连续除极至+80mV,时间为260ms。用O、0.01 nM、1 nM、100 nM、1000 nME2处理H9c2细胞24h后,其平均最大IK分别是(8.19±0.65)(pA/pF)、(4.8±0.52)(pA／pF)、(3.8±0.33)(pA/pF)、(2.87±0.17)(pA／pF)、(2.14±0.13)(pA／pF)。与无E2组相比,0.01 nM、1nM、100 nM、1000 nM E2处理组的平均最大IK有显著性下降(p<0.05)。17β-雌二醇能以浓度依赖方式抑制IK。同样的,17p-雌二醇不改变I-V曲线(n=12-16)。
结论用0、0.01 nM、1 nM、100 nM和1000 nM E2处理H9c2细胞24h后,INa、Ica,L、IK随17β-雌二醇浓度升高而降低。研究表明电压门控钠电流(INa)、L型钙电流(Ica,L)、延迟整流钾电流(Ik)等离子通道电流以浓度依赖的方式随17p-雌二醇浓度升高而降低。
目的研究不同浓度的17p-雌二醇对心肌细胞H9c2细胞形态及损伤程度的影响。
方法Hgc2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,接着分别用荧光探针Fluo-3,SBFI, PBFI,SNARF-1,MQAE孵育细胞,用激光扫描共聚焦显微镜进行细胞形态观察。通过释放到培养基的LDH活性测定和PI染色细胞观察17p-雌二醇对细胞损伤和细胞死亡的影响。
结果将H9c2与不同的荧光探针孵育后,用激光共聚焦显微镜进行形态学观察,发现在无E2或在1000 nM E2时有细胞形态学的改变,细胞变得平而圆。心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000nM17β-雌二醇中培养24小时后,细胞培养基中LDH释放率分别是(11.9±1.1)%,(11.4±1.5)%,(10.2±0.9)%,(9.9±1.6)%,(13.4±1.7)%。无E2组和0.01 nM E2组之间,培养基中LDH释放率无显著性差异(p>0.05),而1nME2组和100 nM E2组中LDH释放率显著低于无E2组和0.01nM E2组(p<0.05)。但1 nM E2和100 nM E2组之间无显著性差异(p>0.05)。1000 nM E2组的H9c2细胞LDH释放率都显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、1 00 nM、1000 nM17β-雌二醇中培养24小时后,细胞死亡率分别是(3.1±0.3)%,(2.9±0.4)%,(2.8±0.5)%,(2.9±0.5)%,(3.8±0.4)%。0.01 nM E2组、1 nM E2组和100 nM E2组细胞细胞死亡率与无E2组相比显著性的下降(p<0.05)。而1 nM E2组和100 nM E2组之间细胞细胞死亡率无显著性差异(p>0.05)。当H9c2细胞用1000 nM E2处理时,其细胞死亡率显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
结论研究表明H9c2细胞在无E2、0.01 nM E2或1000 nM E2条件下,有细胞形态学的改变,并引起细胞损伤和细胞死亡的增加。
During the last century, gender-based differences in the morbidity and mortality of cardiovascular disease have been revealed by epidemiological studies.These studies showed that cardiovascular mortality of pre-menopausal women is significantly smaller than that of men of the same age.Indeed, the risk of cardiovascular disease is increased when the estrogen production stops, either naturally or after surgery or in woman with impaired ovarian function. Cardiovascular disease develops in women on average 10 years later in life compared with men. The decrease of the estrogen level is the main physiological change of the postmenopausal women. On the other hand, experimental studies have shown that natural estrogens, such as 17β-estradiol(E2), can influence cardiovascular disease by acting directly on the heart or by affecting the vascular system. The beneficial effects of estrogen on the development of arteriosclerosis and vascular remodeling are currently under investigation, including its antiapoptotic effects, antioxidant effects, beneficial changes in lipoprotein metabolism, platelet adhesion and aggregation, endothelium-dependent vasodilatation through activation of endothelial nitric oxide synthase (eNOS) with increased expression of eNOS,limiting cardiac hypertrophy and reducing myocardial ischemia. Thus, the differences of morbidity and mortality of cardiovascular disease between men and pre-menopausal women may be due to a protective effect of estrogen.
In contrast to these observations, large randomized clinical trials in postmenopausal women with cardiovascular risk factors or coronary artery disease(CAD), both using conjugated equine estrogens and medroxyprogesterone acetate as hormone "replacement"have recently questioned these atheroprotective effects, because the results showed no effects or even an increase in cardiovascular morbidity and mortality Also, estrogen was reported to increase the risk of heart attack, breast cancer, blood clots, and dementia. These divergent findings resulted in confusion about whether a substitution therapy with natural or novel synthetic sex steroids could represent a therapeutic option for the treatment of atherosclerosis and its complications.One key to solving such an important public health issue would be to better understand the complex mechanisms of acute and chronically administered estrogen action in the cardiovascular system. Therefore, it is important that further studies be carried out to evaluate the beneficial effects of estrogen and its adverse side effects.The effects of estrogen on vascular function have been studied extensively, however, the direct influences of estrogen on heart have received much less consideration. Nowadays, the focus on the beneficial influence of estrogen is gradually shifting from the vascular system to myocardium.It is well known that intracellular ions homeostasis plays an important role in maintaining the functions of the cardiomyocytes. Proper contractile function of the heart depends on ions homeostasis of the cardiac myocytes and intact excitation-contraction. The transient effects of estrogen on single ion channel in the cardiomyocytes have been extensively studied, however, much less is known regarding long term effects of estrogen on several main cardiac ions together. Thus, further studies are required to evaluate the ionic mechanism of estrogen on the cardiomyocytes.
In our previous research, the effect of ovarian sex hormones was investigated using 3-month ovariectomized rat heart.The results of DNA microarray indicated a series of changes in gene expression, including enzymes(Na+,K+-ATPase, Ca2+-ATPase, carbonic anhydrase), ion channel proteins (L-type calcium channel, delayed rectifier potassium channel, voltage-gated sodium channel), exchanger proteins (Na+-H+-exchanger, Na+-Ca2+ exchanger),which were up(down)-regulated more than 2 folds compared with the control.Also, physiological concentration of E2 can enhance gene expression and the activity of Na+,K+-ATPase, sarcoplsmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and carbonic anhydrase(CA) in H9c2 cells. Interestingly, all these proteins or enzymes may associate with communication of electrolytes between intracellular and extracellular apartment. It was therefore hypothesized that physiological concentration of estrogen may maintain normal intracellular ion concentration, acid-base balance and electrophysiological property and play a protective role of estrogen to cardiomyocytes.Otherwise, estrogen may play a completely inverse role on cardiomyocytrs and result in intracellular electrolytes disorder, electrophysiological abnormalities, cardiac injury and even death. In the present, we are attempting to find direct evidence how the estrogen exerts influence on intracellular free ions homeostasis.Using rat myogenic H9c2 cells as an in vitro model,we recently examined intracellular free ion concentrations of Ca2+, Na+, K+ H+ and Cl- and ionic currents of voltage-gated sodium current(INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK).
Objective:To study the long term effect of 17β-estradiol on intracellular free ions concentration of Ca2+,Na+,K+,H+, Cl- in H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, intracellular free ion concentrations([Ca2+]i, [Na+]i, [K+]i, [H+]i, [Cl-]i) were measured by fluorescence assay
Results:When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h,the intracellular free [Ca2+] was (157.5±5.7)nM, (153.8±6.2) nM, (148.5±8.7) nM, (144.5±7.8) nM, (167.5±9.0) nM, respectively. It was demonstrated that the intracellular free calcium ion concentration of H9c2 cells cultured with 0.01 nM,1 nM, or 100 nM 17β-estradiol decreased compared with that in the no E2-treated group(P<0.05).The [Ca2+]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 and 100nM E2(P>0.05).However, [Ca2+]i of H9c2 treated with 1000 nM 17β-estradiol was significantly higher than that treated with no E2,0.01 nM E2,1 nM E2 and 100 nM E2 groups, respectively (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h,the [Na+]i was (8.9±0.5) mM, (8.5±0.8) mM, (7.8±0.8)mM, (7.7±0.9) mM, (9.9±0.7) mM, respectively. The [Na+]i of H9c2 cells treated with 0.01 nM 17β-estradiol was similar to that of no E2 treated group (P>0.05).When H9c2 cells were cultured with 1 nM,100 nM 17β-estradiol,the [Na+]i decreased compared with that of cells treated with no E2 group (P<0.05).The [Na+]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P<0.05).But no significant difference was observed between groups treated with 1 nM and 100 nM E2(P>0.05).Interestingly, [Na+]i of H9c2 incubated with 1000 nM 17β-estradiol was significantly enhanced compared with the no E2 group,0.01 nM E2 group,1 nM E2 group and 100 nM E2 group, respectively(P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [K+] was (134.4±8.3)mM,(137.2±7.5)mM,(140.0±7.5)mM,(142.0±6.8)mM, (133.7±9.6) mM, respectively. The [K+]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated group(P>0.05).The [K+]i in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly higher than that in the group cultured with no E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 nM and 100nM E2 (P>0.05). When H9c2 cells were cultured with 0,0.01 nM, 1000 nM E2 for 24h, the [K+]i decreased compared with that of cells treated with 1 nM E2 (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular pH was (7.0±0.2), (7.3±0.3),(7.4±0.2), (7.4±0.2), (6.4±0.3),respectively. The [pH]i of H9c2 cells treated with 0.01 nM,1 nM,100 nM 17β-estradiol was significantly enhanced compared with no 17β-estradiol group (P<0.05). But there was no significant difference among groups cultured with 0.01 nM,1 nM and 100 nM 17β-estradiol (P>0.05).Also, the [pH]i in H9c2 cells of the group cultured with 1000 nM 17β-estradiol decreased compared that cultured with 0,0.01 nM E2, 1nM E2,100 nM E2 groups, respectively (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [Cl-] was (32.3±3.2)mM,(30.6±4.8)mM,(27.6±4.5)mM,(28.5±4.4)mM, (35.7±4.0) mM, respectively. The [Cl-]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated (P>0.05).The [Cl-]i of H9c2 cells cultured with 1 nM and 100 nM 17β-estradiol decreased compared with that in the no E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 nM and 100 nM E2(P>0.05). However, [Cl"]i in H9c2 cells cultured with 1000 nM increased compared with that in the no E2,0.01 nM E2,1 nM E2 and 100 nM E2 groups, respectively (P<0.05).
Conclusion:The results indicated that 1 nM and 100 nM E2 can maintain the intracellular free ion concentrations of Ca2+,Na+,K+,H+, Cl- in physiological range.No significant difference was observed between groups treated with 1 nM and 100 nM E2 for intracellular free ion concentrations.The treatment of H9c2 cells with no E2,0.01 nM E2 or 1000 nM E2 can disturb the intracellular free ions balance and result in cell injury. It was found that E2 may have important regular role on intracellular free ions homeostasis which is essential to normal function of cardiac cells.
Objective:To study the effect of 17β-estradiol on ionic currents of voltage-gated sodium current (INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK) of H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h.Whole cell patch-clamp technique was used to study ion channel currents (INa, ICa,L, IK) in H9c2 cells.
Results:The voltage dependent activation of INa was measured by 200ms test potential (Vt) that 10mV increased stepwise to 20mV from a holding potential (Vh) of-80mV. This inward current appeared at about-70mV, reached a maximal value around-30mV and reversed at about+20mV. The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100 nM,1000 nM E2 were(3.61±0.42), (2.80±0.29), (2.27±0.30), (2.08±0.35),(1.51±0.20) (pA/pF).The average maximal peak current density in cells cultured with 0.01 nM,1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17p-estradiol suppressed INa in a concentration dependent manner. On the other hand,the 17p-estradiol did not shift the I-V curve (n=12-16/group).
The voltage dependent activation of ICa,L was tested by a 180ms test potential (Vt) that 10mV increased stepwise to 50mV from a holding potential (Vh) of-40mV, reaching a maximal value about OmV.The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100 nM,1000 nM E2 were(9.54±0.75), (7.70±0.81),(6.28±0.62), (5.01±0.38), (4.45±0.57) (pA/pF).Similarly, the average maximal peak current density in cells cultured with 0.01 nM, 1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17β-estradiol suppressed Ica,L in a concentration dependent manner. Similar to the effect on INa, the 17β-estradiol decreased the amplitude of ICa,L without shifting the I-V relationship (n=12-16/group).
IK was induced by 260ms depolarizing pulses from-40mV to +80mV in 10mV steps.The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100nM,1000 nM E2 were(8.19±0.65),(4.80±0.52),(3.81±0.33),(2.87±0.17), (2.14±0.13)(pA/pF).The average maximal peak current density in cells cultured with 0.01 nM,1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17p-estradiol suppressed IK in a concentration dependent manner.Similarly, the 17β-estradiol did not shift the I-V curve (n=12-16/group).
Conclusion Ionic currents of voltage-gated sodium current (INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK) of H9c2 cardiac cells were suppressed via 17β-estradiol in a concentration-dependent manner.
Objective:To study the effect of 17β-estradiol on morphological change, cell injury and cell death of H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, morphological change of the cells was observed by laser scanning confocal microscope.The activity assay of LDH released into the medium and propidium iodide (PI) staining were used to assess the E2 induced cell injury and cell death.
Results:Laser scanning confocal microscope images in H9c2 cells incubated with different fluorescent probes showed that no E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change.The cells became flat and round When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the percent LDH release(% of Max) in the medium was(11.9±1.1), (11.4±1.5),(10.2±0.9), (9.9±1.6),(13.4±1.7), respectively. The percent LDH release in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 group(P>0.05).The percent LDH release in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly decreased compared with the no E2 treated group(P<0.05).There was no significant different between groups cultured with 1 nM and 100 nM E2(P>0.05).Interestingly, the percent LDH release in H9c2 cells cultured with 1000 nM E2 was higher than in the groups cultured with 0,0.01 nM E2,1nM E2 and 100 nM E2 groups, respectively(P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the percent cell death(%) was (3.1±0.3),(2.9±0.4), (2.8±0.5),(2.9±0.5),(3.8±0.4), respectively. The percent cell death in H9c2 cultured with 0.01 nM,1 nM,100 nM E2 decreased compared with that in the no E2 group (P<0.05).No significant difference was observed between groups treated with 1 nM and 100 nM E2(P>0.05).When H9c2 cells were incubated with 1000 nM E2,the percent cell death increased compared with that of cells treated with no E2 group,0.01 nM E2 group,1nM E2 group or 100 nM group(P<0.05).
Conclusion:The treatment of H9c2 cells with no E2,0.01 nM E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change and increase in cell injury, death.
在我们以往的研究中,利用cDNA微阵列技术观察到：去势3个月后,左室心肌细胞的上百个基因的表达发生改变,其中包括Na+,K+-ATP酶、Ca2+-ATP酶、碳酸酐酶、L型钙通道蛋白、延迟整流钾通道蛋白和电压门控钠通道蛋白以及Na+-H+交换蛋白和Na+-Ca2+交换蛋白等的表达。心肌细胞电解质稳定对其功能发挥起至关重要的作用。对心肌细胞H9c2的研究发现生理浓度的雌激素能上调Na+,K+-ATP酶、肌浆网Ca2+-ATP酶和碳酸酐酶的表达和活性,这些酶均可能参与细胞内外电解质平衡的调节。上述的实验结果提示,生理浓度的雌激素水平可能维护心肌细胞内的离子浓度、pH值和膜电生理等特性而保护心肌细胞；否则,会导致电解质紊乱、电生理异常、心肌细胞损伤乃至死亡。因此,本研究的目的是寻找雌激素对心肌细胞内游离离子稳态影响的直接证据。我们用鼠H9c2心肌细胞为研究对象,检测不同浓度雌激素对细胞内Ca2+、Na+、K+、H+和Cl-等离子浓度和ⅠNa、Ⅰca,L、ⅠK等离子电流的影响。
目的研究不同浓度的17β-雌二醇对心肌细胞H9c2内Ca2+、Na+、K+、H+、C1-等游离离子浓度的影响。
方法H9c2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,接着分别用荧光探针Fluo-3,SBFI, PBFI,SNARF-1,MQAE孵育细胞,用荧光法测定细胞内游离离子(Ca2+、Na+、K+、H+、C1-)浓度。
结果心肌细胞H9c2分别在O、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钙离子浓度分别是(157.5±5.7)nM,(153.8±6.2)nM,(148.5±8.7)nM,(144.5±7.8)nM,(167.5±9.0)nM。实验结果显示：与无17β-雌二醇组相比,0.01nM、1 nM和100 nM17β-雌二醇组的细胞内[Ca2+]下降(p<0.05)。无E2组和0.01 nM E2组细胞内[Ca2+]显著高于1 nM E2组(p<0.05)。1 nm和100 nM17p-雌二醇组之间细胞内[Ca2+]无显著性差别(p>0.05),而1000 nM17p-雌二醇组细胞内[Ca2+]却比无E2组和0.01 nME2组,1 nM E2组和100nM E2组有显著性升高(p<0.05)。
心肌细胞H9e2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钠离子浓度分别是(8.9±0.5)mM,(8.5±0.8)mM,(7.8±0.8)mM,(7.7±0.9)mM,(9.9±0.7)mM。0.01 nM17β-雌二醇组细胞内[Na+]与无17p-雌二醇组无显著性差异(p>0.05)。当17p-雌二醇浓度为1 nM、100 nM时,H9c2细胞内[Na+]比无17β-雌二醇时存在显著降低(p<0.05)。无17p-雌二醇组和0.01 nM17β-雌二醇组细胞内[Na+]比1 nM 17p-雌二醇组有显著性升高(p<0.05)。但是1 nM 17β-雌二醇组与100 nnM17β-雌二醇组之间[Na+]无显著性差异(p>0.05)。1000 nM17β-雌二醇组细胞内[Na+]显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离钾离子浓度分别是(134.4±8.3)mM,(137.2±7.5)mM,(140.0±7.5)mM,(142.0±±6.8)mM,(133.7±9.6)mM。对H9c2细胞内的[K+],0.01 nM E2组和无E2组之间无显著性差异(p>0.05)。1 nM和100 nM 2组细胞内[K+]显著性高于无E2组(p<0.05)。1 nM E2和100 nM E2组之间亦无显著性差异(p>0.05)。但是当H9c2细胞分别在O、0.01 nM、1000 nM E2培养24h时,细胞内[K+]比1nM E2组有显著性的下降(p<0.05)。
心肌细胞H9c2分别在0、0.01nM、1nM、100nM、1000nM17p-雌二醇中培养24小时后,心肌细胞内pH分别是(7.0±0.2),(7.3±0.3),(7.44-0.2),(7.44±0.2),(6.44±0.3)。0.01 nM、1 nM、100 nM E2组的H9c2细胞内的pH显著性的高于无E2组(p<0.05)。但是在0.01 nM、1 nM、100 nM E2组之间细胞内pH值无显著性差异(p>0.05)。另外,1000nM E2组的细胞内pH显著性低于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000 nM17p-雌二醇中培养24小时后,心肌细胞内的游离氯离子浓度分别是(32.3±3.2)mM,(30.6±4.8)mM,(27.6±4.5)mM,(28.5±4.4)mM,(35.7±4.0)mM。H9c2细胞内[C1-]在0.01nME2组和无E2组之间无显著性差异(p>0.05)。1 nM E2组和100 nM E2组的[Cl-]i显著性的低于无E2组(p<0.05)而1 nM E2组和100 nM E2之间H9c2细胞内[C1-]无显著性差异(p>0.05)。但是,1000 nM E2组[C1-]分别比无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组显著上升(p<0.05)。
结论研究表明1 nM和100 nM的17p-雌二醇能维持H9c2细胞内Ca2+、Na+、K+、H+、C1-等离子的浓度在生理浓度范围,1 nM和100 nM17p-雌二醇组间细胞内离子浓度无显著性差异。H9c2细胞在无E2、0.01 nME2或1000 nM E2条件下,细胞内游离离子平衡出现紊乱。因此,17p-雌二醇对H9c2细胞内Ca2+、Na+、K+、H+、C1-等游离离子浓度的稳定起着至关重要的调节作用。
目的研究不同浓度的17p-雌二醇对H9c2心肌细胞的电压门控钠电流(INa)、L型钙电流(ICa,L)、延迟整流钾电流(Ik)等离子通道电流的影响。
方法H9c2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,用全细胞膜片钳技术测定H9c2细胞的电压门控钠电流(INa)、L型钙电流(Ica,L)、延迟整流钾电流(Ik)等离子通道电流。
结果电压依赖的INa的测定：维持电位(holding potential)为-80mV,每隔10mV,连续除极至20mV。内向IN。大约在-70mV出现,在-30mV达到最大,而在+20mV逆转。用0、0.01nM、1nM、100 nM、1000nME2处理H9c2细胞24h后,其平均最大INa分别是(3.61±0.42)(pA／pF),(2.80±0.29)(pA/pF),(2.27±0.30)(pA／pF),(2.08±0.31)(pA／pF),(1.51±0.20)(pA／pF)。与无E2组相比,0.01nM、1nM、100nM、1000 nME2处理组的平均最大INa有显著性下降(p<0.05),17β-雌二醇能以浓度依赖的方式抑制INa。另外,17β-雌二醇不改变I-V曲线(n=12-16)。
17β-雌二醇对L型钙电流(Ica,L)的影响维持电位-40mV,每隔10mV连续除极至+50mV,时间180ms。ICa,L在0mV达最大。用O、0.01 nM、1nM、100nM和1000 nME2处理H9c2细胞24h后,其平均最大ICa.L分别是(9.54±0.75)(pA／pF),(7.70±0.81)(pA／pF),(6.28±0.62)(pA／pF),(5.01±0.38)(pA／pF),(4.45±0.57)(pA／pF)。与无E2组相比,0.01 nM、1nM、100 nM、1000 nM E2处理组的平均最大Ica,L有显著性下降(p<0.05)。同样的,17p-雌二醇能以浓度信赖的方式抑制Ica,L。与INa相类似,17p-雌二醇亦不影响I-V关系(n=12-16)。
17β-雌二醇对延迟整流钾电流的影响延迟整流钾电流IK测定：维持电位-40mV,每隔10mV,连续除极至+80mV,时间为260ms。用O、0.01 nM、1 nM、100 nM、1000 nME2处理H9c2细胞24h后,其平均最大IK分别是(8.19±0.65)(pA/pF)、(4.8±0.52)(pA／pF)、(3.8±0.33)(pA/pF)、(2.87±0.17)(pA／pF)、(2.14±0.13)(pA／pF)。与无E2组相比,0.01 nM、1nM、100 nM、1000 nM E2处理组的平均最大IK有显著性下降(p<0.05)。17β-雌二醇能以浓度依赖方式抑制IK。同样的,17p-雌二醇不改变I-V曲线(n=12-16)。
结论用0、0.01 nM、1 nM、100 nM和1000 nM E2处理H9c2细胞24h后,INa、Ica,L、IK随17β-雌二醇浓度升高而降低。研究表明电压门控钠电流(INa)、L型钙电流(Ica,L)、延迟整流钾电流(Ik)等离子通道电流以浓度依赖的方式随17p-雌二醇浓度升高而降低。
目的研究不同浓度的17p-雌二醇对心肌细胞H9c2细胞形态及损伤程度的影响。
方法Hgc2心肌细胞分别在0,0.01 nM,1 nM,100 nM,1000 nM 17p-雌二醇(E2)条件下培养24小时,接着分别用荧光探针Fluo-3,SBFI, PBFI,SNARF-1,MQAE孵育细胞,用激光扫描共聚焦显微镜进行细胞形态观察。通过释放到培养基的LDH活性测定和PI染色细胞观察17p-雌二醇对细胞损伤和细胞死亡的影响。
结果将H9c2与不同的荧光探针孵育后,用激光共聚焦显微镜进行形态学观察,发现在无E2或在1000 nM E2时有细胞形态学的改变,细胞变得平而圆。心肌细胞H9c2分别在0、0.01 nM、1 nM、100 nM、1000nM17β-雌二醇中培养24小时后,细胞培养基中LDH释放率分别是(11.9±1.1)%,(11.4±1.5)%,(10.2±0.9)%,(9.9±1.6)%,(13.4±1.7)%。无E2组和0.01 nM E2组之间,培养基中LDH释放率无显著性差异(p>0.05),而1nME2组和100 nM E2组中LDH释放率显著低于无E2组和0.01nM E2组(p<0.05)。但1 nM E2和100 nM E2组之间无显著性差异(p>0.05)。1000 nM E2组的H9c2细胞LDH释放率都显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
心肌细胞H9c2分别在0、0.01 nM、1 nM、1 00 nM、1000 nM17β-雌二醇中培养24小时后,细胞死亡率分别是(3.1±0.3)%,(2.9±0.4)%,(2.8±0.5)%,(2.9±0.5)%,(3.8±0.4)%。0.01 nM E2组、1 nM E2组和100 nM E2组细胞细胞死亡率与无E2组相比显著性的下降(p<0.05)。而1 nM E2组和100 nM E2组之间细胞细胞死亡率无显著性差异(p>0.05)。当H9c2细胞用1000 nM E2处理时,其细胞死亡率显著性的高于无E2组,0.01 nM E2组,1 nM E2组和100 nM E2组(p<0.05)。
结论研究表明H9c2细胞在无E2、0.01 nM E2或1000 nM E2条件下,有细胞形态学的改变,并引起细胞损伤和细胞死亡的增加。
During the last century, gender-based differences in the morbidity and mortality of cardiovascular disease have been revealed by epidemiological studies.These studies showed that cardiovascular mortality of pre-menopausal women is significantly smaller than that of men of the same age.Indeed, the risk of cardiovascular disease is increased when the estrogen production stops, either naturally or after surgery or in woman with impaired ovarian function. Cardiovascular disease develops in women on average 10 years later in life compared with men. The decrease of the estrogen level is the main physiological change of the postmenopausal women. On the other hand, experimental studies have shown that natural estrogens, such as 17β-estradiol(E2), can influence cardiovascular disease by acting directly on the heart or by affecting the vascular system. The beneficial effects of estrogen on the development of arteriosclerosis and vascular remodeling are currently under investigation, including its antiapoptotic effects, antioxidant effects, beneficial changes in lipoprotein metabolism, platelet adhesion and aggregation, endothelium-dependent vasodilatation through activation of endothelial nitric oxide synthase (eNOS) with increased expression of eNOS,limiting cardiac hypertrophy and reducing myocardial ischemia. Thus, the differences of morbidity and mortality of cardiovascular disease between men and pre-menopausal women may be due to a protective effect of estrogen.
In contrast to these observations, large randomized clinical trials in postmenopausal women with cardiovascular risk factors or coronary artery disease(CAD), both using conjugated equine estrogens and medroxyprogesterone acetate as hormone "replacement"have recently questioned these atheroprotective effects, because the results showed no effects or even an increase in cardiovascular morbidity and mortality Also, estrogen was reported to increase the risk of heart attack, breast cancer, blood clots, and dementia. These divergent findings resulted in confusion about whether a substitution therapy with natural or novel synthetic sex steroids could represent a therapeutic option for the treatment of atherosclerosis and its complications.One key to solving such an important public health issue would be to better understand the complex mechanisms of acute and chronically administered estrogen action in the cardiovascular system. Therefore, it is important that further studies be carried out to evaluate the beneficial effects of estrogen and its adverse side effects.The effects of estrogen on vascular function have been studied extensively, however, the direct influences of estrogen on heart have received much less consideration. Nowadays, the focus on the beneficial influence of estrogen is gradually shifting from the vascular system to myocardium.It is well known that intracellular ions homeostasis plays an important role in maintaining the functions of the cardiomyocytes. Proper contractile function of the heart depends on ions homeostasis of the cardiac myocytes and intact excitation-contraction. The transient effects of estrogen on single ion channel in the cardiomyocytes have been extensively studied, however, much less is known regarding long term effects of estrogen on several main cardiac ions together. Thus, further studies are required to evaluate the ionic mechanism of estrogen on the cardiomyocytes.
In our previous research, the effect of ovarian sex hormones was investigated using 3-month ovariectomized rat heart.The results of DNA microarray indicated a series of changes in gene expression, including enzymes(Na+,K+-ATPase, Ca2+-ATPase, carbonic anhydrase), ion channel proteins (L-type calcium channel, delayed rectifier potassium channel, voltage-gated sodium channel), exchanger proteins (Na+-H+-exchanger, Na+-Ca2+ exchanger),which were up(down)-regulated more than 2 folds compared with the control.Also, physiological concentration of E2 can enhance gene expression and the activity of Na+,K+-ATPase, sarcoplsmic/endoplasmic reticulum Ca2+-ATPase (SERCA) and carbonic anhydrase(CA) in H9c2 cells. Interestingly, all these proteins or enzymes may associate with communication of electrolytes between intracellular and extracellular apartment. It was therefore hypothesized that physiological concentration of estrogen may maintain normal intracellular ion concentration, acid-base balance and electrophysiological property and play a protective role of estrogen to cardiomyocytes.Otherwise, estrogen may play a completely inverse role on cardiomyocytrs and result in intracellular electrolytes disorder, electrophysiological abnormalities, cardiac injury and even death. In the present, we are attempting to find direct evidence how the estrogen exerts influence on intracellular free ions homeostasis.Using rat myogenic H9c2 cells as an in vitro model,we recently examined intracellular free ion concentrations of Ca2+, Na+, K+ H+ and Cl- and ionic currents of voltage-gated sodium current(INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK).
Objective:To study the long term effect of 17β-estradiol on intracellular free ions concentration of Ca2+,Na+,K+,H+, Cl- in H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, intracellular free ion concentrations([Ca2+]i, [Na+]i, [K+]i, [H+]i, [Cl-]i) were measured by fluorescence assay
Results:When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM, 100 nM and 1000 nM 17β-estradiol for 24 h,the intracellular free [Ca2+] was (157.5±5.7)nM, (153.8±6.2) nM, (148.5±8.7) nM, (144.5±7.8) nM, (167.5±9.0) nM, respectively. It was demonstrated that the intracellular free calcium ion concentration of H9c2 cells cultured with 0.01 nM,1 nM, or 100 nM 17β-estradiol decreased compared with that in the no E2-treated group(P<0.05).The [Ca2+]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 and 100nM E2(P>0.05).However, [Ca2+]i of H9c2 treated with 1000 nM 17β-estradiol was significantly higher than that treated with no E2,0.01 nM E2,1 nM E2 and 100 nM E2 groups, respectively (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h,the [Na+]i was (8.9±0.5) mM, (8.5±0.8) mM, (7.8±0.8)mM, (7.7±0.9) mM, (9.9±0.7) mM, respectively. The [Na+]i of H9c2 cells treated with 0.01 nM 17β-estradiol was similar to that of no E2 treated group (P>0.05).When H9c2 cells were cultured with 1 nM,100 nM 17β-estradiol,the [Na+]i decreased compared with that of cells treated with no E2 group (P<0.05).The [Na+]i in H9c2 cells cultured with no E2 or 0.01 nM E2 was significantly higher than that in the group cultured with 1 nM E2 treated group(P<0.05).But no significant difference was observed between groups treated with 1 nM and 100 nM E2(P>0.05).Interestingly, [Na+]i of H9c2 incubated with 1000 nM 17β-estradiol was significantly enhanced compared with the no E2 group,0.01 nM E2 group,1 nM E2 group and 100 nM E2 group, respectively(P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [K+] was (134.4±8.3)mM,(137.2±7.5)mM,(140.0±7.5)mM,(142.0±6.8)mM, (133.7±9.6) mM, respectively. The [K+]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated group(P>0.05).The [K+]i in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly higher than that in the group cultured with no E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 nM and 100nM E2 (P>0.05). When H9c2 cells were cultured with 0,0.01 nM, 1000 nM E2 for 24h, the [K+]i decreased compared with that of cells treated with 1 nM E2 (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular pH was (7.0±0.2), (7.3±0.3),(7.4±0.2), (7.4±0.2), (6.4±0.3),respectively. The [pH]i of H9c2 cells treated with 0.01 nM,1 nM,100 nM 17β-estradiol was significantly enhanced compared with no 17β-estradiol group (P<0.05). But there was no significant difference among groups cultured with 0.01 nM,1 nM and 100 nM 17β-estradiol (P>0.05).Also, the [pH]i in H9c2 cells of the group cultured with 1000 nM 17β-estradiol decreased compared that cultured with 0,0.01 nM E2, 1nM E2,100 nM E2 groups, respectively (P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the intracellular free [Cl-] was (32.3±3.2)mM,(30.6±4.8)mM,(27.6±4.5)mM,(28.5±4.4)mM, (35.7±4.0) mM, respectively. The [Cl-]i in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 treated (P>0.05).The [Cl-]i of H9c2 cells cultured with 1 nM and 100 nM 17β-estradiol decreased compared with that in the no E2 treated group(P<0.05).There was no significant difference between groups cultured with 1 nM and 100 nM E2(P>0.05). However, [Cl"]i in H9c2 cells cultured with 1000 nM increased compared with that in the no E2,0.01 nM E2,1 nM E2 and 100 nM E2 groups, respectively (P<0.05).
Conclusion:The results indicated that 1 nM and 100 nM E2 can maintain the intracellular free ion concentrations of Ca2+,Na+,K+,H+, Cl- in physiological range.No significant difference was observed between groups treated with 1 nM and 100 nM E2 for intracellular free ion concentrations.The treatment of H9c2 cells with no E2,0.01 nM E2 or 1000 nM E2 can disturb the intracellular free ions balance and result in cell injury. It was found that E2 may have important regular role on intracellular free ions homeostasis which is essential to normal function of cardiac cells.
Objective:To study the effect of 17β-estradiol on ionic currents of voltage-gated sodium current (INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK) of H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h.Whole cell patch-clamp technique was used to study ion channel currents (INa, ICa,L, IK) in H9c2 cells.
Results:The voltage dependent activation of INa was measured by 200ms test potential (Vt) that 10mV increased stepwise to 20mV from a holding potential (Vh) of-80mV. This inward current appeared at about-70mV, reached a maximal value around-30mV and reversed at about+20mV. The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100 nM,1000 nM E2 were(3.61±0.42), (2.80±0.29), (2.27±0.30), (2.08±0.35),(1.51±0.20) (pA/pF).The average maximal peak current density in cells cultured with 0.01 nM,1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17p-estradiol suppressed INa in a concentration dependent manner. On the other hand,the 17p-estradiol did not shift the I-V curve (n=12-16/group).
The voltage dependent activation of ICa,L was tested by a 180ms test potential (Vt) that 10mV increased stepwise to 50mV from a holding potential (Vh) of-40mV, reaching a maximal value about OmV.The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100 nM,1000 nM E2 were(9.54±0.75), (7.70±0.81),(6.28±0.62), (5.01±0.38), (4.45±0.57) (pA/pF).Similarly, the average maximal peak current density in cells cultured with 0.01 nM, 1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17β-estradiol suppressed Ica,L in a concentration dependent manner. Similar to the effect on INa, the 17β-estradiol decreased the amplitude of ICa,L without shifting the I-V relationship (n=12-16/group).
IK was induced by 260ms depolarizing pulses from-40mV to +80mV in 10mV steps.The average maximal peak current density of H9c2 cells in the groups cultured with 0,0.01 nM,1nM,100nM,1000 nM E2 were(8.19±0.65),(4.80±0.52),(3.81±0.33),(2.87±0.17), (2.14±0.13)(pA/pF).The average maximal peak current density in cells cultured with 0.01 nM,1 nM,100 nM, or 1000 nM E2 decreased compared with that in the no E2 group(p<0.05).17p-estradiol suppressed IK in a concentration dependent manner.Similarly, the 17β-estradiol did not shift the I-V curve (n=12-16/group).
Conclusion Ionic currents of voltage-gated sodium current (INa), L-type calcium current (ICa,L) and delayed rectifier potassium current (IK) of H9c2 cardiac cells were suppressed via 17β-estradiol in a concentration-dependent manner.
Objective:To study the effect of 17β-estradiol on morphological change, cell injury and cell death of H9c2 cardiac cells.
Methods:H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM,1000 nM E2 for 24h. Then H9c2 cells were incubated with fluorescent probes, morphological change of the cells was observed by laser scanning confocal microscope.The activity assay of LDH released into the medium and propidium iodide (PI) staining were used to assess the E2 induced cell injury and cell death.
Results:Laser scanning confocal microscope images in H9c2 cells incubated with different fluorescent probes showed that no E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change.The cells became flat and round When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the percent LDH release(% of Max) in the medium was(11.9±1.1), (11.4±1.5),(10.2±0.9), (9.9±1.6),(13.4±1.7), respectively. The percent LDH release in H9c2 cells treated with 0.01 nM E2 was similar to that of no E2 group(P>0.05).The percent LDH release in H9c2 cells cultured with 1 nM and 100 nM E2 was significantly decreased compared with the no E2 treated group(P<0.05).There was no significant different between groups cultured with 1 nM and 100 nM E2(P>0.05).Interestingly, the percent LDH release in H9c2 cells cultured with 1000 nM E2 was higher than in the groups cultured with 0,0.01 nM E2,1nM E2 and 100 nM E2 groups, respectively(P<0.05).
When H9c2 cardiac cells were cultured with 0,0.01 nM,1 nM,100 nM and 1000 nM 17β-estradiol for 24 h, the percent cell death(%) was (3.1±0.3),(2.9±0.4), (2.8±0.5),(2.9±0.5),(3.8±0.4), respectively. The percent cell death in H9c2 cultured with 0.01 nM,1 nM,100 nM E2 decreased compared with that in the no E2 group (P<0.05).No significant difference was observed between groups treated with 1 nM and 100 nM E2(P>0.05).When H9c2 cells were incubated with 1000 nM E2,the percent cell death increased compared with that of cells treated with no E2 group,0.01 nM E2 group,1nM E2 group or 100 nM group(P<0.05).
Conclusion:The treatment of H9c2 cells with no E2,0.01 nM E2 or 1000 nM E2 can resulted in H9c2 cardiac cells morphological change and increase in cell injury, death.
引文
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76. Hanke H, Kamenz J, Hanke S.Effect of 17-beta estradiol on pre-existing atherosclerotic lesions:role of the endothelium. Atherosclerosis.1999, 147:123-132.
77. Rosenfeld ME, Kauser K, Martin-McNulty B, et al.Estrogen inhibits the initiation of fatty streaks throughout the vasculature but does not inhibit intra-plaque hemorrhage and the progression of established lesions in apolipoprotein E deficient mice. Atherosclerosis,2002,164:251-259.
78.Wagner JD, Clarkson TB.The applicability of hormonal effects on atherosclerosis in animals to heart disease in postmenopausal women. Semin Reprod Med,2005,23:149-156.
79.Herrington DM, Espeland MA, Crouse JR, et al.Estrogen replacement and brachial artery flow-mediated vasodilation in older women. Arterioscler Thromb Vasc Biol,2001,21:1955-1961.
80.Dubey RK, Imthurn B, Barton M, et al.Vascular consequences of menopause and hormone therapy:importance of timing of treatment and type of estrogen.. Cardiovasc Res,2005,66:295-306.
81.Lehtimaki T, Dastidar P, Jokela H, et al.Effect of long-term hormone
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82.Makela R, Dastidar P, Jokela H, et al.Effect of long-term hormone replacement therapy on atherosclerosis progression in postmenopausal women relates to myeloperoxidase promoter polymorphism. J Clin Endocrinol Metab,2003, 88:3823-3828.
83.Herrington DM, Howard TD, Hawkins GA, et al.Estrogen-receptor polymorphisms and effects of estrogen replacement on high-density lipoprotein cholesterol in women with coronary disease. N Engl J Med,2002,346:967-974.
84. Shearman AM, Cupples LA, Demissie S,et al. Association between estrogen reportor alpha gene variation and cardiovascular disease.JAMA,2003, 290:2263-2270.
85.Grodstein F, Clarkson TB, Manson JE, et al. Understanding the divergent data on postmenopausal hormone therapy. N Engl J Med,2003,348:645-650.
86.Koh KK, Shin MS,Sakuma I, et al.Effects of conventional or lower doses of hormone replacement therapy in postmenopausal women. Arterioscler Thromb Vasc Biol,2004,24:1516-1521.
87.Ferrara A, Quesenberry CP, Karter AJ, et al.Current use of unopposed estrogen and estrogen plus progestin and the risk of acute myocardial infarction among women with diabetes:the Northern California Kaiser Permanente Diabetes Registry,1995-1998.Circulation,2003,107:43-48.
88.Gomes MP, Deitcher SR. Risk of venous thromboembolic disease associated with hormonal contraceptives and hormone replacement therapy:a clinical review. Arch Intern Med,2004,164:1965-1976.
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