醛固酮於心房心肌細胞中透過CREB增加納鈣交換體之表現:在心房震顫中醛固酮之角色含義

Abstract

心房震顫是所有心律不整症狀中最為常見的一種，且有相當高的發病率及死亡率。在近幾十年中，許多研究指出腎素-血管張力素-醛固酮系統(rennin-angiotensin-aldosterone system;RAAS)對心房震顫扮演著非常重要的角色，在過去實驗室的研究中發現到經由胞外電位紀錄在HL-1新房心肌細胞中，Angiotensin II (AngII)可以透過鈉鈣交換蛋白 (sodium-calcium exchanger; NCX)及cAMP-response element binding protein (CREB)造成震顫現象。另一方面，aldsoterone也被證實會去誘發心律不整的產生。在本篇研究中，我們想要了解與AngII同屬於RAAS的aldosterone是否會去調控NCX mRNA表現，且也想去了解CREB是否也參與在其中的訊息傳遞。我們發現同樣在不同濃度或不同時間的醛固酮刺激下，NCX mRNA的表現量有隨之上升的情形。同樣也在NCX的基因預測出promoter中有CREB的binding site，利用染色質免疫共沉澱法，確認了CREB的確可以與所預測的結合位做結合。除此之外，同樣利用轉染dominant-negative CREB(pCMV-CREB133和pCMV-KCREB)到HL-1中發現到因醛固酮所造成的NCX mRNA表現量上升的情況因此減弱。最後的實驗發現醛固酮所造成的CREB磷酸化程度的上升並不會因為給予了PKC inhibitor或PKD inhibitor而有所減弱，但再給予蛋白質生成抑制劑(cycloheximide)、礦物質皮質固酮接受器阻斷劑(mineralocorticoid receptor blocker; MR blocker)或活性氧物質清除劑(ROS scavenger; N-acetyl cysteine)後醛固酮所誘導的CREB磷酸化增加則有下降的情形。綜合上述，醛固酮可以增加NCX mRNA的表現量，同時醛固酮可以透過ROS或MR的genomic effect進而增加CREB磷酸化的程度

Atrial fibrillation (AF) is a common arrhythmia that causes significant morbidity and mortality. In recent decades, the rennin-angiotensin-aldosterone system (RAAS) has been proven to play an important role in AF. Previous results in our lab have demonstrated that angiotensin II can induce fibrillary-like electrical activity in HL-1 atrail myocytes through sodium-calcium exchanger (NCX) and cAMP response element-binding protein (CREB). Aldosterone has also been shown to trigger AF. In this study, we investigated whether aldosterone, a critical component in RAAS, can modulate the expression of NCX mRNA and whether CREB is involved in its signaling pathway. We found that aldosterone increased NCX expression through a concentration and time dependent manner. A CREB binding site was identified in the promoter of NCX gene, and binding of CREB to this cognate binding site was confirmed by chromatin-immunoprecipitation assay (ChIP). We further found that aldosterone increased CREB serine 133 phosphorylation, and aldosterone induced NCX expression was attenuated by dominant-negative CREB (pCMV-CREB133 and pCMV-KCREB). Finally, aldosterone induced CREB serine 133 phosphorylation was not inhibited by protein kinase C or protein kinase D inhibitor, but was attenuated by protein synthesis inhibitor cycloheximide, mineralocorticoid receptor blocker spironolactone, and reactive oxygen species scavenger N-acetyl cysteine. In conclusion, aldosterone increases NCX expression thorugh CREB, and aldosterone increases CREB phosphorylation through reactive oxygen species and the genomic effect of mineralocorticoid receptor.