雌激素及雌激素接受體β抑制ISO誘導心肌細胞肥大與凋亡之機轉

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Title:	雌激素及雌激素接受體β抑制ISO誘導心肌細胞肥大與凋亡之機轉 E2/ERβ ameliorate ISO-induced myocardial cell hypertrophic and apoptotic effects
Authors:	劉建男;Chien-Nan Liu
Contributors:	醫學院基礎醫學研究所
Keywords:	雌激素;雌激素接受體;心臟肥大;心肌凋亡;estrogen;estrogen receptor beta;cardiac hypertrophy;myocardial cell apoptosis
Date:	2010
Issue Date:	2010-09-29 12:13:34 (UTC+8)
Abstract:	先前的研究中指出，女性罹患心血管疾病的機率遠比男性低，然而隨著年紀的增加，女性則失去此優勢，此現象或許與雌激素分泌減少有關。同時已知壓力及交感神經過度興奮刺激，可導致心衰竭，而交感神經接受體及蛋白磷酸酶，例如 calcineurin(PP2B)與PP1確實參與調控心臟肥大與凋亡的機轉與路徑。雖然目前已知，雌激素可藉由雌激素接受體抑制心臟衰竭，達到保護心臟的功能。然而，雌激素與雌激素接受體 β作用機轉不明，加上estrogen與estrogen receptor β保護心肌的機轉是否藉由影響交感神經接受體及蛋白磷酸酶PP2B及PP1，來調控心肌功能仍是未知。在第一部分的實驗結果，我們首先確認ISO (isoproterenol)誘發心肌肥大相關ANP及BNP蛋白大量表現，同時誘發上游調控蛋白CaMKII、calcineurin、GATA4及NFAT的活化及細胞外型大小的增加。進而，在actin染色法發現，雌激素與雌激素接受體 β均可抑制ISO所誘導的心肌細胞肥大的現象。在核質分離的實驗中更發現，雌激素與雌激素接受體 β可抑制ISO所誘導之NFAT轉錄因子進核現象。同時其下游的心肌肥大指標BNP蛋白也有相同調控情形。接著，我們採用calcineurin抑制劑(CsA)在H9c2及初代培養心肌細胞實驗中均發現，calcineurin確實扮驗雌激素及雌激素接受體 β抑制ISO誘發心肌細胞肥大的重要中間者角色。最後，從鈣離子螯合劑的實驗中更發現，雌激素與雌激素接受體 β確實是藉由調降ISO誘導之鈣離子內流而抑制calcineurin調控蛋白的活性。進而活化I-1蛋白且抑制PP1，使PLB磷酸化且活化，使得促進心肌細胞質內鈣離子回收至胞漿網SR (Sarcoplasmic Reticulum)中。雌激素接受體抑制劑更證明雌激素是經由雌激素接受體 β進而抑制calcineurin的活性，而達到抑制calcineurin/NFAT 肥大訊息路徑，使得ISO所誘導之心肌肥大現象得以減緩。從第二部分的實驗結果，我們從TUNEL assay中發現，雌激素與雌激素接受體 β可抑制ISO (isoproterenol)所誘導的心肌細胞凋亡現象。在粒線體分離的實驗中發現，可看到ISO誘導之釋放型cytochrome C及粒線體中之Bad蛋白均被雌激素與雌激素接受體 β抑制。同時，其下游caspase 3活性也可見得同樣的情形。接著，我們同時在H9c2及初代培養心肌細胞實驗中發現，雌激素與雌激素接受體 β抑制ISO所誘導之calcineurin蛋白，且主要是經由促進其蛋白降解。在實驗中我們更進一步地發現，雌激素與雌激素接受體 β是藉由促進Akt磷酸化，並使MDM2活化，經PI3K/Akt/MDM2路徑而促進calcineurin蛋白的降解，來達到抑制經由ISO誘導之calcineurin所造成的心肌細胞凋亡作用。總結實驗結果，雌激素可經由雌激素接受體 β抑制ISO所誘導的鈣離子內流及calcineurin活化。是經由促進I-1蛋白活性且抑制PP1，使PLB磷酸化來促進肌漿網鈣離子的回收，降低ISO誘導的心肌肥大現象。同時，雌激素及雌激素接受體 β可經由Akt活化與MDM2磷酸化而造成calcineurin蛋白降解，進而抑制ISO所誘導的心肌細胞凋亡。我們的實驗建議了雌激素及大量表現雌激素接受體 β之基因治療方式，應可阻斷壓力所造成之心肌細胞傷害。 Heart disease is markedly higher in men than in women. However, this advantage of women decline with age increase, the malfunction of estrogen secretion and estrogen receptor might be one of the reasons. 17β-Estradiol (E2) has been reported to prevent development of cardiac hypertrophy and apoptosis via estrogen receptors (ERs). In addition, protein phosphatase, such as protein phosphatase 1 (PP1) and calcineurin (PP2B) are involved with cardiac hypertrophy and cell apoptosis signaling. However, the mechanisms of E2/ER β to suppress cardiac hypertrophy and apoptosis are not fully understood, and the roles protein phosphatase in E2/ERβ action is waiting for further investigation. From the results of first part, we confirmed that ISO-induced cardiohypertrophic related proteins, ANP and BNP protein levels upregulated the upstream regulation proteins, p-CaMKII, calcineurin, p-GAT4, NFATc3 activation, resulted in the increase of H9c2 cardiomyoblast cell size. Moreover, we found that E2/ERβ inhibit ISO-induced H9c2 cell hypertrophy in actin staining assay. In addition, E2/ ERβ inhibit ISO-induced NFAT translocation in nuclear extraction assay, and attenuated protein level of downstream marker, BNP. Moreover, we applied the calcineurin inhibitor (CsA) in H9c2 and neonatal rat ventricular myocyte (NRVM), and found that calcineurin plays as the key mediator for E2/ERβ function . At last, the cells treated with calcium blocker BATPA totally blocked the E2/ERβ inhibit ISO-induced Ca2+ influx and hypertrophic effects, which suggested E2/ERβ inhibit calcineurin activity, to activate I-1 protein and suppress PP1, then induce PLB protein phosphorylation and activation, resulted in the Ca2+ reuptake into sarcoplasmic reticulum through SR Ca2+ cycling modification. Our data indicated that E2/ERβ suppress the Ca2+ influx and calcineurin activity induced by ISO to enhance the PLB protein activity and SR Ca2+ cycling. From the results of second part, we observed that E2/ERβ inhibit the ISO -induced myocardial cell apoptosis in TUNEL assay, the cytochrome C released in mitochondria isolation assay and the downstream apoptotic marker caspase 3 activation. Moreover, we found E2/ERβ block ISO-induced H9c2 cell apoptosis through the enhancement of calcineurin protein degradation. At last, we further identified E2/ERβ enhance calcineurin degradation through the PI3K/Akt/MDM2 signaling pathway, to inhibit ISO-induced myocardial cell apoptosis. Taken together, E2/ERβ attenuate ISO-induced myocardial cell hypertrophy through inhibited Ca2+ influx and calcineurin activation, resulted in I-1 activation, PP1 suppression and PLB activation, which induce the Ca2+ re-uptaked by SR. In the mean time, E2/ERβ activate the PI3K/Akt/MDM2 pathway to induce calcineurin protein degradation, which block the effect of ISO-induced myocardial cell apoptosis effect. Our research suggests that estrogen supplement and/or estrogen receptor β gene overexpression therapy might be good ways for stress-induced myocardial damage treatment.
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