本文選題：心肌肥大 + 內(nèi)質(zhì)網(wǎng)應(yīng)激�。� 參考：《山東大學》2010年博士論文

【摘要】： 心肌肥大是心肌細胞對多種病理因素的代償性反應(yīng),但慢性心肌肥大參與了擴張性心肌病、心力衰竭、猝死等多種心血管疾病的發(fā)生、發(fā)展過程,嚴重危及人類生命；因此闡明心肌肥大的發(fā)病機制對于防治心肌肥大和心力衰竭具有重要的臨床意義。 內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum, ER)是調(diào)節(jié)細胞內(nèi)鈣穩(wěn)態(tài)和膜型／分泌型蛋白質(zhì)合成、折疊的重要細胞器,對維持心肌細胞鈣和功能蛋白質(zhì)穩(wěn)態(tài)具有重要作用。ER對應(yīng)激刺激非常敏感,多種因素如鈣紊亂、蛋白合成增加、缺血、缺氧等因素均可觸發(fā)ER應(yīng)激。適當?shù)腅R應(yīng)激誘導ER分子伴侶如葡萄糖調(diào)節(jié)蛋白(glucose-regulated proteins, GRPs)、鈣網(wǎng)蛋白(calreticulin, CRT)等表達上調(diào),增強ER鈣調(diào)節(jié)和處理未折疊蛋白的能力,具有細胞保護作用；但是,持續(xù)或嚴重的ER應(yīng)激可觸發(fā)ER凋亡信號通路,促進ER重要的促凋亡蛋白C/EBP homologous protein (CHOP)表達增加和caspase-12剪切活化,導致細胞凋亡和組織損傷。蛋白激酶R樣ER激酶(protein kinase R-like ER kinase, PERK)是重要的ER跨膜蛋白,ER應(yīng)激時GRP78與PERK的解離激活PERK,誘導激活型轉(zhuǎn)錄因子4 (activating transcription factor 4, ATF4)轉(zhuǎn)錄及其下游靶分子CHOP表達,促進細胞凋亡。 多種證據(jù)表明ER應(yīng)激與神經(jīng)退行性變、糖尿病性心肌病和缺血／再灌注損傷等多種疾病密切相關(guān)。研究發(fā)現(xiàn)心肌肥大時ER應(yīng)激分子CRT、GRP78、GRP94等表達顯著上調(diào),CHOP介導的ER應(yīng)激凋亡通路參與了心力衰竭過程中的細胞凋亡,提示心肌肥大過程中存在ER應(yīng)激反應(yīng),但ER應(yīng)激在心肌肥大發(fā)生、發(fā)展中的變化及作用目前并不清楚。心肌細胞內(nèi)Ca2+水平升高參與了心肌肥大的發(fā)生,是觸發(fā)心肌肥大反應(yīng)的基本信號。鈣調(diào)神經(jīng)磷酸酶(calcineurin, CaN)是一種受細胞漿Ca2+及鈣調(diào)素(calmodulin, CaM)調(diào)控的蛋白絲氨酸／蘇氨酸磷酸酶,由催化亞基CnA和調(diào)節(jié)亞基CnB組成,在T細胞活化、心肌肥大、細胞周期調(diào)控等多種生物學過程中發(fā)揮關(guān)鍵性的調(diào)控作用。心肌細胞增強因子2c (myocyte enhancer factor 2c, MEF2c)是MEF2c家族中發(fā)現(xiàn)的第一個參與心臟發(fā)育的分子,并被CaN激活,但是CaN-MEF2c信號途徑在ER應(yīng)激誘導心肌肥大中的作用目前尚未報道。 本工作首先在大鼠腹主動脈狹窄致高血壓、心肌肥大模型上,探討ER應(yīng)激在心肌肥大發(fā)生、發(fā)展過程中的作用；其次采用ER應(yīng)激誘導劑毒胡蘿卜素(thapsigargin, TG,抑制ER鈣泵繼而排空ER內(nèi)Ca2+)和衣霉素(tunicamycin, TM,抑制ER內(nèi)蛋白質(zhì)N-端糖基化)作用于原代培養(yǎng)乳鼠心肌細胞,觀察TG和TM對心肌細胞肥大和ER應(yīng)激的影響,并探討CaN-MEF2c信號途徑在ER應(yīng)激誘導心肌細胞肥大過程中的作用。主要實驗方法和結(jié)果如下： 1 ER應(yīng)激參與大鼠腹主動脈狹窄致高血壓心肌肥大發(fā)生、發(fā)展過程 本部分實驗旨在觀察ER應(yīng)激反應(yīng)在腹主動脈狹窄致高血壓大鼠心肌肥大發(fā)生、發(fā)展過程中的作用。健康雄性Wistar大鼠85只,隨機分為模型組(n=45)和假手術(shù)組(n=40),模型組行腹主動脈狹窄術(shù),假手術(shù)組僅分離腹主動脈不行狹窄術(shù),分別于術(shù)后1 d、3 d、7 d、14 d、28 d時觀察各組血流動力學變化,測定全心重／體重比(whole heart weight/body weight, HW/BW)和左心室重／體重比(left ventricular weight/body weight, LVW/BW),雙向電泳-質(zhì)譜分析技術(shù)檢測術(shù)后28 d心肌組織蛋白質(zhì)表達譜的變化,RT-PCR技術(shù)檢測左心室心肌組織ER應(yīng)激分子GRP78、CRT和CHOP等mRNA表達變化,Western blot分析α-肌動蛋白(α-actin)、GRP78. CRT、CHOP,以及凋亡相關(guān)蛋白Bax和Bcl-2等表達變化。 結(jié)果發(fā)現(xiàn)腹主動脈狹窄誘導大鼠心肌肥大,與假手術(shù)組比較,術(shù)后7d模型組大鼠血壓升高,心功能代償性增加,HW/BW和LVW/BW顯著增加。狹窄術(shù)后28 d心肌肥大標志性蛋白肌球蛋白輕鏈表達上調(diào),而心臟a-actin前體和α-肌球蛋白重鏈表達下調(diào),Western blot證實模型組心肌組織a-actin表達較假手術(shù)組顯著增加。其次發(fā)現(xiàn)模型組CRT mRNA表達于術(shù)后1d即發(fā)生顯著上調(diào),較假手術(shù)組增加136%(P0.01)；GRP78表達于術(shù)后7d顯著增加,其高表達均持續(xù)至實驗結(jié)束。長期ER應(yīng)激觸發(fā)CHOP凋亡途徑,模型組大鼠心肌組織CHOP和促凋亡蛋白Bax表達均于術(shù)后14d顯著增加,而抗凋亡蛋白Bcl-2表達降低。上述結(jié)果提示腹主動脈狹窄早期即可觸發(fā)ER應(yīng)激,誘導ER分子伴侶表達增加,ER應(yīng)激反應(yīng)可能參與了腹主動脈狹窄致大鼠高血壓、心肌肥大過程。CHOP介導的ER應(yīng)激相關(guān)凋亡途徑可能參與了心肌肥大及失代償?shù)恼{(diào)節(jié),決定肥大心肌失代償?shù)倪M程。2 ER應(yīng)激誘導劑TG和TM誘導原代培養(yǎng)心肌細胞肥大和顯著ER應(yīng)激 上述實驗發(fā)現(xiàn)ER應(yīng)激參與了腹主動脈狹窄致大鼠心肌肥大的發(fā)生、發(fā)展過程,為證實ER應(yīng)激獨立誘導心肌肥大,本部分實驗在原代培養(yǎng)乳鼠心肌細胞模型上,采用不同濃度TG(1、2.5、5、10、50、70、100 nmol／L)處理原代培養(yǎng)心肌細胞48 h或50 nmol／LTG分別處理心肌細胞12、24、36、48、60、72 h；同時采用不同濃度TM(1、10、100 ng／m1)分別處理心肌細胞48、72、96 h；10-7mmol／L血管緊張素(angiotensin, Ang)Ⅱ處理心肌細胞48 h作為陽性對照。采用培養(yǎng)基乳酸脫氫酶(lactate dehydrogenase, LDH)活性和細胞凋亡率檢測反映心肌細胞損傷變化,RT-PCR技術(shù)觀察心肌細胞肥大標志性基因心房鈉尿肽(atrial natriuretic peptide, ANP)和腦鈉肽(brain natriuretic peptide, BNP) mRNA表達,3H-亮氨酸([3H]-Leucine)摻入技術(shù)檢測心肌細胞蛋白質(zhì)合成速率,F-actin染色技術(shù)觀測心肌細胞骨架改變,同時分析心肌細胞表面積變化。此外采用RT-PCR技術(shù)觀測ER應(yīng)激分子CRT、GRP78、PERK、ATF4和CHOP mRNA表達,Western blot技術(shù)觀測CRT、GRP78、CHOP、Bax和Bel-2蛋白水平改變,同時采用ER特異性熒光染料Dapoxyl觀察ER形態(tài)變化,免疫熒光技術(shù)觀察CRT熒光改變。 結(jié)果發(fā)現(xiàn),ER應(yīng)激誘導劑TG和TM以時間和劑量依賴性方式誘導心肌細胞損傷,培養(yǎng)基LDH活性和細胞凋亡率顯著增加；同時心肌細胞顯著肥大,表現(xiàn)為TG和TM以時間和劑量依賴性方式誘導ANP和BNP mRNA表達、蛋白合成速率和細胞表面積增加；F-actin染色表明TG和TM誘導心肌細胞骨架熒光強度顯著增強、應(yīng)力纖維增加。而且發(fā)現(xiàn)50 nmol／L TG作用48 h和10 ng／ml TM作用72 h心肌細胞顯著肥大,細胞損傷較輕,是誘導心肌細胞肥大的較適條件。此外,TG以劑量和時間依賴性方式誘導培養(yǎng)心肌細胞顯著ER應(yīng)激,ER應(yīng)激分子CRT、GRP78表達顯著增加；值得注意的是PERK和ATF4 mRNA表達于TG作用24-48 h顯著增加,而在60-72 h時顯著降低。嚴重ER應(yīng)激觸發(fā)細胞凋亡途徑,TG以劑量和時間依賴性方式促進ER凋亡蛋白CHOP表達增加和Bcl-2/Bax比值顯著降低。心肌細胞ER染色顯示ER形態(tài)顯著擴張,熒光顆粒分布不均、濃集并出現(xiàn)空泡。免疫熒光顯示TG作用后CRT熒光強度增強且向核周濃集。10 ng／ml TM處理72 h心肌細胞也證實上述ER應(yīng)激改變。上述結(jié)果提示ER應(yīng)激誘導劑TG和TM誘導培養(yǎng)心肌細胞顯著肥大和ER應(yīng)激,而且CHOP凋亡途徑激活參與了ER應(yīng)激誘導的心肌細胞肥大。3 CaN-MEF2c信號途徑參與ER應(yīng)激誘導的心肌細胞肥大過程 ER是細胞內(nèi)重要的Ca2+處理器,細胞內(nèi)Ca2+升高是觸發(fā)心肌細胞肥大的基本信號,而CaN可直接受細胞內(nèi)Ca2+調(diào)控,本部分實驗旨在探討CaN-MEF2c信號途徑在ER應(yīng)激誘導心肌細胞肥大過程中的作用。不同濃度TG(1、2.5、5、10、50、70、100 nrnol／L)處理原代培養(yǎng)心肌細胞48 h或50 nmol／L TG分別處理心肌細胞12、24、36、48、60、72 h,同時采用10ng／ml TM處理心肌細胞72 h。其次為觀察CaN活性抑制后心肌細胞肥大和ER應(yīng)激變化,在50 nmol／L TG作用48 h誘導心肌細胞肥大模型上,采用CaN活性抑制劑環(huán)孢素A(CsA,5μmol/L)預(yù)處理心肌細胞10 min后繼而培養(yǎng)基內(nèi)加入50nmol/LTG作用48 h。采用Fluo-3AM染色檢測心肌細胞內(nèi)游離Ca2+水平,超微量Ca2+-ATP酶活性測試盒檢測肌漿網(wǎng)／內(nèi)質(zhì)網(wǎng)鈣-ATPase(sarco／ER Ca2+-ATPase,SERCA)活性,同時采用底物發(fā)色法檢測心肌細胞CaN活性,采用細胞凋亡率和培養(yǎng)基LDH活性檢測反映CaN活性抑制后細胞損傷情況,采用ANP和BNP mRNA表達、蛋白合成速率和細胞表面積檢測評價CaN活性抑制后心肌細胞肥大變化。Western blot技術(shù)檢測心肌細胞SERCA,受磷蛋白(phospholamban, PLB)、MEF2c、p-MEF2c以及CaN活性抑制后ER應(yīng)激分子CRT、GRP78、CHOP、Bax和Bcl-2等蛋白表達,免疫熒光技術(shù)檢測MEF2c熒光改變。 結(jié)果發(fā)現(xiàn)ER應(yīng)激誘導劑TG以劑量和時間依賴性方式誘導細胞內(nèi)游離Ca2+水平升高,同時SERCA活性和表達降低、PLB表達增加；此外心肌細胞CaN活性和MEF2c/p-MEF2c蛋白表達顯著增加。TM作用心肌細胞也證實ER應(yīng)激誘導心肌細胞內(nèi)游離Ca2+水平增加和SERCA活性降低,同時CaN活性升高。免疫熒光顯示正常心肌細胞MEF2c主要分布在細胞漿,TG作用后MEF2c向核內(nèi)轉(zhuǎn)位。CsA顯著抑制TG誘導的心肌細胞CaN活性升高,同時阻斷TG誘導的心肌細胞肥大,表現(xiàn)為CsA顯著阻斷TG誘導的心肌細胞ANP、BNP mRNA表達、蛋白質(zhì)合成速率和細胞表面積增加。免疫熒光發(fā)現(xiàn)TG誘導的心肌細胞MEF-2c表達和核轉(zhuǎn)位均顯著被CsA抑制。心肌細胞肥大抑制后細胞損傷顯著增加,細胞凋亡率和培養(yǎng)基LDH活性升高。此外發(fā)現(xiàn)CaN活性抑制沒有阻斷TG誘導的CRT、GRP78和CHOP表達增加。上述結(jié)果提示SERCA活性降低和PLB表達增加可能參與了心肌細胞內(nèi)游離Ca2+增加,Ca2+可能通過激活CaN-MEF2c信號途徑參與ER應(yīng)激誘導的心肌細胞肥大過程。心肌細胞肥大可能是ER應(yīng)激損傷的代償性反應(yīng),CaN-MEF2c途徑阻斷顯著抑制ER應(yīng)激誘導的心肌細胞肥大,導致細胞損傷增加,至少部分是通過CHOP介導的ER應(yīng)激凋亡途徑實現(xiàn)的。 通過上述分析,得出如下實驗結(jié)論：ER應(yīng)激不僅參與腹主動脈狹窄致高血壓大鼠心肌肥大的發(fā)生、發(fā)展過程,而且獨立誘導培養(yǎng)乳鼠心肌細胞肥大發(fā)生,CaN-MEF2c信號途徑參與了ER應(yīng)激誘導的心肌細胞肥大發(fā)生、發(fā)展過程,CaN活性抑制顯著阻斷ER應(yīng)激誘導的心肌細胞肥大,同時細胞損傷顯著增加。提示ER應(yīng)激是心肌肥大的發(fā)病學因素之一,為心肌肥大、心力衰竭的臨床治療提供了新的靶點。
[Abstract]:Myocardial hypertrophy is a compensatory response to a variety of pathological factors, but chronic myocardial hypertrophy is involved in the occurrence and development of many kinds of cardiovascular diseases, such as dilated cardiomyopathy, heart failure, sudden death and so on, and seriously endangers human life. Therefore, it is important to clarify the pathogenesis of myocardial hypertrophy for the prevention and treatment of cardiac hypertrophy and heart failure. The clinical significance.
Endoplasmic reticulum (ER) is an important organelle that regulates intracellular calcium homeostasis and membrane / secretory protein synthesis and folds. It plays an important role in maintaining calcium and functional protein homeostasis in cardiac myocytes..ER is very sensitive to stress stimulation. Many factors such as calcium disorder, protein synthesis, ischemia, and hypoxia can be touched. ER stress. Appropriate ER stress induces the up regulation of ER molecular chaperones such as glucose-regulated proteins (GRPs), calreticulin (calreticulin, CRT) and so on, enhancing the ability of ER calcium regulation and processing unfolded proteins to protect the cells. However, the persistent or severe ER stress triggers the ER apoptotic signaling pathway. The important apoptotic protein C/EBP homologous protein (CHOP) expression increases and caspase-12 shear activation, leading to cell apoptosis and tissue damage. Protein kinase R like ER kinase (protein kinase R-like ER) is an important transmembrane protein. Vating transcription factor 4 (ATF4) transcripts and its downstream target molecule CHOP expression promotes cell apoptosis.
A variety of evidence suggests that ER stress is closely related to neurodegenerative changes, diabetic cardiomyopathy and ischemia / reperfusion injury. The expression of ER stress molecules CRT, GRP78, GRP94 is significantly up-regulated during myocardial hypertrophy, and CHOP mediated ER stress apoptosis pathway participates in apoptosis during cardiac failure and suggests myocardial hypertrophy. There is a ER stress reaction in the process, but the changes and roles of ER stress in cardiac hypertrophy are not clear. The increase of Ca2+ levels in cardiac myocytes is the basic signal to trigger cardiac hypertrophy. Calcineurin (CaN) is a kind of cytoplasmic Ca2+ and calmodulin (CALMODUL). In, CaM) regulated protein serine / threonine phosphatase, composed of catalytic subunit CnA and regulatory subunit CnB, plays a key role in many biological processes, such as T cell activation, cardiac hypertrophy, cell cycle regulation, and so on. Cardiomyocyte enhancement factor 2C (myocyte enhancer factor 2C, MEF2c) is the first found in the MEF2c family. A molecule that participates in cardiac development and is activated by CaN, but the role of CaN-MEF2c signaling pathway in ER stress induced cardiac hypertrophy has not been reported.
In this work, we first discussed the role of ER stress in the development and development of cardiac hypertrophy in hypertensive rat abdominal aortic stenosis and myocardial hypertrophy, followed by ER stress inducer (thapsigargin, TG, inhibition of ER calcium pump and emptying ER Ca2+) and ycomycin (tunicamycin, TM, N- terminal sugar in ER). The effects of TG and TM on cardiomyocyte hypertrophy and ER stress were observed and the effect of CaN-MEF2c signal pathway on the hypertrophy of cardiomyocytes induced by ER stress was investigated. The main experimental methods and results were as follows:
1 ER stress participates in the occurrence and development of hypertensive cardiac hypertrophy induced by abdominal aortic stenosis in rats.
This part of the experiment was designed to observe the role of ER stress reaction in the development of hypertrophy of myocardium in hypertensive rats induced by abdominal aorta stenosis. 85 healthy male Wistar rats were randomly divided into model group (n=45) and sham operation group (n=40), the model group underwent abdominal aorta stenosis, and the sham operation group was only separated from abdominal aorta stenosis. After 1 D, 3 D, 7 d, 14 d, and 28 d, the hemodynamic changes were observed in each group. The changes of the total cardiac weight / weight ratio (whole heart weight/body weight, HW/BW) and the left ventricular weight / weight ratio (left ventricular) were measured, and the changes in the protein expression profiles of 28 cardiac muscle tissues were detected by two dimensional electrophoresis mass spectrometry. The changes in the expression of ER stress molecules, such as GRP78, CRT and CHOP, were detected in left ventricular myocardium, and Western blot was used to analyze alpha actin (alpha -actin), GRP78. CRT, CHOP, and the expression of apoptosis related proteins.
The results showed that the abdominal aorta stenosis induced the hypertrophy of the rat myocardium. Compared with the sham operation group, the blood pressure of the 7d model group was increased, the cardiac function was increased and the HW/BW and LVW/BW increased significantly. The expression of the myosin light chain expression of the 28 d cardiac hypertrophy was up, and the expression of the a-actin precursor and the alpha myosin heavy chain expression was down, W Estern blot confirmed that the expression of a-actin in the myocardial tissue of the model group was significantly higher than that of the sham operation group. Secondly, it was found that the expression of CRT mRNA in the model group was significantly up-regulated after the operation and increased by 136% (P0.01) than that of the sham operation group. The expression of GRP78 in the model group increased significantly after the operation, and the high expression of the GRP78 was increased to the end of the experiment. Long term ER stress triggered CHOP apoptotic pathway and model. The expression of CHOP and apoptotic protein Bax in myocardial tissue of group rats increased significantly after operation, but the expression of anti apoptotic protein Bcl-2 decreased. The results suggested that ER stress could be triggered at the early stage of abdominal aorta stenosis and the expression of ER molecular chaperone increased, and ER stress reaction may be involved in hypertension in rats caused by abdominal aorta stenosis and.CH of cardiac hypertrophy. OP mediated ER stress related apoptosis pathway may be involved in the regulation of myocardial hypertrophy and decompensation, determining the process of hypertrophic cardiomyocyte decompensation,.2 ER stress inducer TG and TM induced primary cultured cardiomyocytes hypertrophy and significant ER stress
These experiments showed that ER stress was involved in the occurrence of myocardial hypertrophy in rats with abdominal aorta stenosis. The development process was that ER stress was independent to induce myocardial hypertrophy independently. In this part of this experiment, the primary cultured rat cardiomyocyte model was treated with different concentrations of TG (1,2.5,5,10,50,70100 nmol / L) to treat the primary cultured cardiomyocytes 48 h or 50 nmol / LTG. 12,24,36,48,60,72 h of cardiac myocytes was treated with TM (1,10100 ng / M1) at the same time, 48,72,96 h in cardiac myocytes was treated respectively. 10-7mmol / L angiotensin (angiotensin, Ang) II was used to treat cardiac myocytes as positive control. The activity of culture based lactate dehydrogenase and the rate of apoptosis were used. The detection of myocardial cell damage changes, RT-PCR technique was used to observe the expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (brain natriuretic peptide, BNP) mRNA, and 3H- leucine ([3H]-Leucine) technique was used to detect the protein synthesis rate of cardiac myocytes. RT-PCR technique was used to observe the expression of ER stress molecules CRT, GRP78, PERK, ATF4 and CHOP mRNA. Western blot technique was used to observe CRT, GRP78, protein and protein levels. Detection of CRT fluorescence changes.
The results showed that ER stress inducer TG and TM induced cardiomyocyte injury in time and dose dependent manner, the activity of LDH and the rate of apoptosis increased significantly, and the cardiac myocytes were significantly hypertrophic, which showed that TG and TM induced ANP and BNP mRNA form with time and dose dependent manner, and the protein synthesis rate and cell surface area increased; F increased. -actin staining showed that the fluorescence intensity of the cytoskeleton induced by TG and TM was significantly enhanced and the stress fibers were increased. Moreover, 50 nmol / L TG action was found in 48 h and 10 ng / ml TM, and the 72 h myocardial cells were significantly hypertrophy, and the cell damage was lighter. Furthermore, TG induced the culture heart in a dose and time dependent manner. The expression of CRT and GRP78 in the muscle cells was significantly increased by ER stress, and the expression of ER stress molecules CRT and GRP78 was significantly increased. It was worth noting that the expression of PERK and ATF4 mRNA was significantly increased in the action of TG, but decreased significantly at the time of 60-72 H. The ER staining showed that the ER morphology was significantly expanded, the distribution of fluorescent particles was uneven, and the vacuoles appeared. The fluorescence intensity of CRT was enhanced after the immunofluorescence of TG and the concentration of.10 ng / ml TM to 72 h cardiomyocytes was also confirmed. The above-mentioned results suggested that ER stress inducer TG and induce the culture of myocardium Significant cell hypertrophy and ER stress, and the activation of CHOP apoptosis pathway involved in ER induced cardiomyocyte hypertrophy.3 CaN-MEF2c signaling pathway involved in ER induced hypertrophy of cardiomyocytes.
ER is an important Ca2+ processor in cells. The increase of Ca2+ in cells is the basic signal to trigger cardiac myocyte hypertrophy, and CaN can be directly regulated by intracellular Ca2+. This part of the experiment aims to explore the role of CaN-MEF2c signal pathway in the process of cardiac hypertrophy induced by ER stress. The different concentration TG (1,2.5,5,10,50,70100 nrnol / L) deals with the original generation. Cardiac myocytes were treated with 48 h or 50 nmol / L TG respectively, and 12,24,36,48,60,72 h was treated respectively with 10NG / ml TM treatment of cardiac myocytes 72 h. followed by the observation of cardiomyocyte hypertrophy and ER stress changes after the inhibition of CaN activity. 5 mol/L) the pretreated cardiomyocytes were followed by 10 min and cultured in the culture base to add 50nmol/LTG to 48 h.. Fluo-3AM staining was used to detect the level of free Ca2+ in cardiac myocytes. Ultra micro Ca2+-ATP enzyme activity test box was used to detect the activity of sarcoplasmic reticulum / endoplasmic reticulum calcium -ATPase (sarco / ER Ca2+-ATPase, SERCA) activity, and the substrate chromophore was used to detect cardiac myocytes Activity, cell apoptosis rate and culture medium LDH activity were used to detect cell damage after inhibition of CaN activity, ANP and BNP mRNA were used, protein synthesis rate and cell surface area were detected to evaluate the hypertrophy of cardiomyocytes after CaN activity inhibition,.Western blot technique was used to detect cardiac myocyte SERCA, phosphoprotein (phospholamban, PLB), MEF2c, MEF2c and CaN activity inhibited ER stress molecules CRT, GRP78, CHOP, Bax and Bcl-2 protein expression, and immunofluorescence technique was used to detect MEF2c fluorescence changes.
The results showed that ER stress inducer TG induced the increase of intracellular free Ca2+ level in dose and time dependent manner, while SERCA activity and expression decreased, and PLB expression increased. In addition, the CaN activity and the expression of MEF2c/p-MEF2c protein in cardiac myocytes significantly increased.TM action myocardial cells also confirmed the increase of free Ca2+ level in cardiac myocytes induced by ER stress. The activity of addition and SERCA decreased and the activity of CaN increased. The immunofluorescence showed that MEF2c in normal cardiac myocytes was mainly distributed in the cytoplasm. After the action of TG, the transposition of MEF2c to the nucleus.CsA significantly inhibited the increase of CaN activity induced by TG, while blocking TG induced cardiomyocyte hypertrophy. The expression of CsA significantly blocked TG induced cardiomyocyte ANP. Expression of protein synthesis rate and cell surface area increased. Immunofluorescence showed that the expression of MEF-2c and nuclear transposition of TG induced cardiomyocytes were significantly inhibited by CsA. The cell damage was significantly increased after the inhibition of myocardial hypertrophy, and the apoptosis rate and the activity of LDH were increased. Furthermore, the inhibition of CaN activity did not block CRT, GRP78 and CH induced by TG. The above results suggest that the decrease of SERCA activity and the increase of PLB expression may participate in the increase of intracellular free Ca2+ in cardiac myocytes. Ca2+ may participate in ER stress induced cardiomyocyte hypertrophy by activating CaN-MEF2c signal pathway. Cardiomyocyte hypertrophy may be a compensatory response to ER stress injury, and CaN-MEF2c pathway blocking is significantly inhibited. ER stress induced cardiomyocyte hypertrophy, resulting in increased cellular damage, at least partly through CHOP mediated ER stress pathway.
Based on the above analysis, the following conclusions are drawn: ER stress is not only involved in the occurrence and development of cardiac hypertrophy in hypertensive rats induced by abdominal aortic stenosis.

【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R363

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