| 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 assayResults: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.
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