Estrogen Regulates The Expression Of Na～+, K～+-ATPase β1 Subunit, Sarcoplasmic Reticulum Ca～(2+)-ATPase And Carbonic Anhydrase IV

Backgrounds: Cardiovascular disease is the first killer of thehuman health. Many factors can increase the attack rate ofcardiovascular disease, and the most dangerous factor is age. The attackrate of cardiovascular disease in young women is lower than men withthe same age, and the high risk age of women is 10-15 years later thanmen. When women get in menopause stage, the risk of coronary heartdisease increases 3-4 folders than before to the level which is same asthe that of men with the same age. The typical change of the menopausalwomen is the significant decrease of estrogen. Now there are intensedebates in whether the estrogen replacement therapy (ERT) is benefit tothe menopausal women. In the year 2005, Science, Mendelssohn andKaras argued that estrogen has protection effect to heart, and it isnecessary to improve the biological understanding of the perimenopauseand gender differences in cardiovascular disease, and elucidate theeffects of E2 on heart at the molecular and cellular level.The former viewpoint of the protect role of estrogen to heart isfocuses on the "indirect protection". Now the subject whether estrogenhas "direct protection" become the hot spot. In our former study, weestablished three SD rat models of sham, ovariectomy and estrogenreplacement treatment. The results of ischemia/reperfusion experiments indicated that there was no pathological change of the aeteria coronariain ovariectomied rat, but the descent of heart function in ovariectomiedrat was observed. The results of DNA micro array indicated serious geneexpression changes including Na⁺,K⁺-ATPaseβ1 subunit,sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase 2a (SERCA2a) andcarbonic anhydraseâ…£(CAâ…£). Na⁺, K⁺-ATPase, SERCA and CAâ…£arefundamental elements in maintaining the normal internal environment ofthe heart cells. These results suggested that the decrease of estrogen inmenopausal women may alter the expression of certain proteins andresult in the changes of dielectric concentration, pH and theelectrophysiological characteristics of the heart cells. In this article, weintend to illuminate the regulation role of estrogen using rat embryonicheart-derived H9c2 cells.Part 1 The regulation role of estrogen on the activity ofNa⁺, K⁺-ATPase, SERCA and the expression of Na⁺, K⁺-ATPaseβ1subunit, SERCA2a and CAâ…£Methods: Rat embryonic heart H9c2 cells were cultured in a tissueflask(at 5% CO₂) containing DMEM supplemented with 10% fetal calfserum. The serum was stripped of E2 using dextran T-70-coated activated charoal. The single-handed effect of E2 on H9c2 cells wasexamined as follows. For long-term effect of E2, cells were incubatedfor 24 h with 0, 0.01 nM, 1 nM, 100 nM, or 1μM E2 before experiments.To understand the short-term effects of E2, the cells were incubated with0, 1 nM or 100 nM E2 for 5 min, 10 min, 20 min and 30 min. Thegroups treated without E2 served as controls.Results: The activity of Na⁺, K⁺-ATPase in H9c2 cells treated with0.01 nM E2 was similar to that of control (p＞0.05). The activity ofNa⁺,K⁺-ATPase in H9c2 cells cultured with 1 nM E2 was significantlyenhanced compared with that in the group cultured with 0.01 nM E2 andthe control group (p＜0.05, p＜0.05). The activity of the group culturedwith 100 nM E2 was significantly higher than that cultured with 0.01nM E2 and the control group (p＜0.05, p＜0.05). There was nosignificant difference between groups cultured with 1 nM and 100 nME2 (p＞0.05). No significant difference was observed between groupstreated with 1μM and 1 nM E2 (p＞0.05). The activity of SERCA incells cultured with 0.01 nM, 1 nM, 100 nM and 1μM E2 increasedcompared with the control group (p＜0.05, p＜0.05, p＜0.05, p＜0.05).The activity of SERCA in cells cultured with 1 nM and 100 nM E2 washigher than that in the group cultured with 0.01 nM E2 (p＜0.05, p＜0.05). When H9c2 cells were incubated with 1μM E2, the activity ofSERCA decreased compared with that of the cells treated with 100 nM E2 (p＜0.05). No significant difference was observed between groupstreated with 1μM and 1 nM E2(p＞0.05).The mRNA level of Na⁺, K⁺-ATPaseβ1 subunit in H9c2 cellscultured with 1 nM and 100 nM E2 increased compared with cellstreated with 0.01 nM E2 and control(p＜0.05). No significant differencewas observed between the former two groups. The mRNA expression ofNa⁺, K⁺-ATPaseβ1 subunit in cells treated with 1μM E2 wassignificantly lower than that in cells cultured with 100 nM E2 (p＜0.05),and was similar to that of the 1 nM E2 group (p＞0.05).For SERCA2a, the expression was significantly up-regulated in thegroups treated with 0.01 nM, 1 nM and 100 nM E2 compared withcontrol(p＜0.05, p＜0.05, p＜0.05), and a significant difference wasobserved among these three groups (p＜0.05). The expression ofSERCA2a decreased in the group treated with 1μM E2 compared withthe group treated with 100 nM E2 (p＜0.05).The mRNA expression of CAâ…£under 1 nM and 100 nM E2 washigher than that in the group cultured with 0.01 nM E2 and control(p＜0.05, p＜0.05), and there was no significant difference between theformer two groups (p＞0.05). The mRNA expression of CAâ…£in thegroup cultured with 1μM E2 was similar to the cells cultured with 100nM E2 (p＞0.05), and higher than the expression of the cells culturedwith 0.01 nM E2 (p＜0.05). The tendency of expression changes of protein levels ofNa⁺, K⁺-ATPaseβ1 subunit, SERCA2a and CAâ…£in different E2concentration groups is consistent with the results of RT-PCR.Conclusion: 1. Estrogen can enhance the activity ofNa⁺,K⁺-ATPase and SERCA, up-regulate the expression of Na⁺, K⁺-ATPaseβ1 subunit, SERCA2a and CAâ…£on mRNA and protein level. 2.A dose-dependent effect of E2 on the activities of Na⁺, K⁺-ATPase andSERCA was observed in this study, and the shape of the E2concentration-enzymic activity curve was similar to that of thetemperature-enzymic activity curve. 3. It was demonstrated that E2regulated the activity of Na⁺,K⁺-ATPase and SERCA through geneexpression but not the short-term effect pathway. 4. Present studysuggested that estrogen has the direct protection effect to heart, and thedose of E2 and treatment cycle used in HRT is important in menopausalwomen.Part 2. The study on the mechanisms of estrogen in regulating theexpression of Na⁺,K⁺-ATPaseβ1 subunit, SERCA2a and CAâ…£Methods: H9c2 cells were cultured for 24 h with 1 nM E2, 1 nME2 plus 1 nM tamoxifen or 1 nM E2 plus 10 nM tamoxifen (TAM). Thecells cultured with 1 nM E2 served as controls. To investigate the effect of tamoxifen, cells were treated with 10 nM tamoxifen alone for 24 h.Results: The activity of Na⁺,K⁺-ATPase in cells cultured with 1 nME2 plus 1 nM tamoxifen or 1 nM E2 plus 10 nM tamoxifen was notsignificantly different compared with the control (p＞0.05, p＞0.05),but a decreasing tendency was observed with increasing tamoxifenconcentration. The expression of Na⁺, K⁺-ATPaseβ1 subunit in the cellscultured with 1 nM E2 plus 10 nM tamoxifen was significantly lowerthan that in the control group (p＜0.05). No significant difference wasobserved between the group treated with 1 nM E2 plus 1 nM tamoxifenand the control group (p＞0.05).The activity of SERCA and the expression of SERCA2a at mRNAand protein levels in H9c2 cells cultivated with 1 nM E2 plus 10 nMtamoxifen was significantly lower than that in cells treated with 1 nM E2(p＜0.05), and higher than that in the 0 nM E2 group (p＜0.05). Nosignificant difference was observed between the groups treated 1 nM E2plus 1 nM tamoxifen and the control group.Along with the upgraded tamoxifen concentration, a decreasedtendency of expression of CAâ…£at mRNA and protein levels wasobserved, but there was no significant difference(p＞0.05).Tamoxifen alone had no significant effect on these proteinscompared with the group treated with 0 nM E2(p＞0.05).Conclusion: The results indicates that estrogen receptors might be involved in the regulation of Na⁺,K⁺-ATPaseβ1 subunit, SERCAexpression induced by E2. Other signaling pathways may be involved inthe up-regulation of CAâ…£expression induced by E2, and deservesfurther study.