| ObjectiveL-type calcium channels （Ca-L）, are widely found in the membranes of variousexcitable cells, such as skeletal muscle cell, cardiomyocyte, cardiovascular smoothmuscle cell, endocrine cell, neurons cell. It plays an extremely important role in theformation and maintenance of the action potential plateau, and also the initiation ofexcited-contraction coupling in cardiomyocyte. Change in the Ca-L function isknown as the important factor to affect early afterdepolarization （EAD） and causetorsade de pointes （TdP）. Researches previous have shown that estrogen is animportant factor affecting the TdP susceptibility, and also can upregulate theexpression of Cav1.21C, the main functional subunit of Ca-L, via a altering geneexpression way. The purpose of this study is to further explore the transductionmechanism of estrogen regulation on Ca-L.Method（1） After incubated with estrogen or other reagents, the level of Cav1.21C proteinin primary myocardial cells or cells of cell line H9C2was measured by Western blot.（2） Incubated with E2or other reagents, the activity of nuclear transcription factor-κB（NF-κB）, and the phosphorylation of cAMP response element binding protein（p-CREB） of primary myocardial cells or cells of cell line H9C2was detected byELISA or Western blot.（3） Using the transfection reagent oligofectamine to thetransfect the CREB-decoy oligonucleotide into the cells, and then incubated withestrogen, the Cav1.21C protein level was measured by Western blot.（4） Singlemyocardial cells of rabbits were isolated by enzymic method. According to the designof our experiment, cardiomyocytes or cells of H9C2were incubated with estrogen or other reagents. ICa-L。was recorded by whole-cell patch clamp technique in thevoltage-clamp mode.Result（1） Estrogen （1100nM） incubation for24h caused an increase of ICa-L。density （p<0.05） and Cav1.21C expression （p <0.05） in cells of H9C2via a dose dependentmanner. As the same, Estrogen （100nM） incubation for24h also caused an increasein Cav1.21C expression （p <0.05）; while the upregulation of Cav1.21C wasblocked when treated with estrogen receptor antagonist ICI182,780.（2） Estrogen （100nM） incubation for30min could significantly increase theexpression of p-CREB （p <0.05）, but with the extension of time, p-CREB activitydecreased. In addition, Estrogen could inhibit the activity of the NF-κB （p <0.05）.（3） Compared to the Estrogen treatment group, Cav1.21C expression was lowerwhen cells of H9C2were incubated with PI3K inhibitor LY294002and estrogen （p <0.05）; but MAPK U0126, and PKA inhibitor KT5720treatment didn’t decreaseCav1.21C expression enhanced by E2in H9C2cells. Results from Western blotshowed that, Cav1.21C expression was lower than E2treatment （p <0.05）, whenincubated with both E2and PI3K inhibitor （p <0.05）; but MAPK U0126, and PKAinhibitor KT5720treatment didn’t decrease Cav1.21C expression enhanced by E2inH9C2cells. While whole-cell patch clamp result from rabbit cardiomyocytesshowed that, PI3K inhibitor （LY294002） and PKA inhibitor （KT5720） couldsignificantly suppress estrogen-induced enhancement of ICa-L。（p <0.05）, but U0126had no significant impact.（4） PI3K inhibitor LY294002co-incubated with estrogen inhibits estrogen-inducedincreased expression of p-CREB （p <0.05）.（5） After transfected control decoy nucleotide, estrogen could still increaseCav1.21C expression in cells of H9C2（p <0.01）; while CREB-decoyoligonucleotide could significantly decrease estrogen-induced enhancement of Cav1.21C （p <0.01）.ConclusionEstrogen showed a dose-dependent and receptor dependent upregulation of L-typecalcium channel and ICa-L。in rat cardiomyocyte. Estrogen could also significantlyincrease the activity of CREB, but significantly inhibit the activity of NF-κB. PI3Kinhibitor could significantly suppress estrogen-induced enhancement of Cav1.21C inH9C2cells, but MAPK and PKA inhibitor didn’t have such effect. While results inrabbit cardiomyocytes showed that, PI3K inhibitor and PKA inhibitor couldsignificantly inhibit the estrogen-induced enhancement of cardiac ICa-L。, but MAPKinhibit didn’t have such effect. PI3K inhibitor could significantly suppressestrogen-induced CREB activity in H9C2cells. CREB-decoy oligonucleotide couldsignificantly suppress estrogen-induced enhancement of Cav1.21C. From the resultsabove, we can conclude that estrogen may upregulate the Cav1.21C expressionthrough the estrogen receptor（ER）/PI3K/CREB pathway in H9C2cells. |
