CALCINEURIN-MEF2C信号途径参与内质网应激诱导的大鼠心肌细胞肥大
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摘要
心肌肥大是心肌细胞对多种病理因素的代偿性反应,但慢性心肌肥大参与了扩张性心肌病、心力衰竭、猝死等多种心血管疾病的发生、发展过程,严重危及人类生命；因此阐明心肌肥大的发病机制对于防治心肌肥大和心力衰竭具有重要的临床意义。
内质网(endoplasmic reticulum, ER)是调节细胞内钙稳态和膜型／分泌型蛋白质合成、折叠的重要细胞器,对维持心肌细胞钙和功能蛋白质稳态具有重要作用。ER对应激刺激非常敏感,多种因素如钙紊乱、蛋白合成增加、缺血、缺氧等因素均可触发ER应激。适当的ER应激诱导ER分子伴侣如葡萄糖调节蛋白(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)等表达上调,增强ER钙调节和处理未折叠蛋白的能力,具有细胞保护作用；但是,持续或严重的ER应激可触发ER凋亡信号通路,促进ER重要的促凋亡蛋白C/EBP homologous protein (CHOP)表达增加和caspase-12剪切活化,导致细胞凋亡和组织损伤。蛋白激酶R样ER激酶(protein kinase R-like ER kinase, PERK)是重要的ER跨膜蛋白,ER应激时GRP78与PERK的解离激活PERK,诱导激活型转录因子4 (activating transcription factor 4, ATF4)转录及其下游靶分子CHOP表达,促进细胞凋亡。
多种证据表明ER应激与神经退行性变、糖尿病性心肌病和缺血／再灌注损伤等多种疾病密切相关。研究发现心肌肥大时ER应激分子CRT、GRP78、GRP94等表达显著上调,CHOP介导的ER应激凋亡通路参与了心力衰竭过程中的细胞凋亡,提示心肌肥大过程中存在ER应激反应,但ER应激在心肌肥大发生、发展中的变化及作用目前并不清楚。心肌细胞内Ca2+水平升高参与了心肌肥大的发生,是触发心肌肥大反应的基本信号。钙调神经磷酸酶(calcineurin, CaN)是一种受细胞浆Ca2+及钙调素(calmodulin, CaM)调控的蛋白丝氨酸／苏氨酸磷酸酶,由催化亚基CnA和调节亚基CnB组成,在T细胞活化、心肌肥大、细胞周期调控等多种生物学过程中发挥关键性的调控作用。心肌细胞增强因子2c (myocyte enhancer factor 2c, MEF2c)是MEF2c家族中发现的第一个参与心脏发育的分子,并被CaN激活,但是CaN-MEF2c信号途径在ER应激诱导心肌肥大中的作用目前尚未报道。
本工作首先在大鼠腹主动脉狭窄致高血压、心肌肥大模型上,探讨ER应激在心肌肥大发生、发展过程中的作用；其次采用ER应激诱导剂毒胡萝卜素(thapsigargin, TG,抑制ER钙泵继而排空ER内Ca2+)和衣霉素(tunicamycin, TM,抑制ER内蛋白质N-端糖基化)作用于原代培养乳鼠心肌细胞,观察TG和TM对心肌细胞肥大和ER应激的影响,并探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。主要实验方法和结果如下：
1 ER应激参与大鼠腹主动脉狭窄致高血压心肌肥大发生、发展过程
本部分实验旨在观察ER应激反应在腹主动脉狭窄致高血压大鼠心肌肥大发生、发展过程中的作用。健康雄性Wistar大鼠85只,随机分为模型组(n=45)和假手术组(n=40),模型组行腹主动脉狭窄术,假手术组仅分离腹主动脉不行狭窄术,分别于术后1 d、3 d、7 d、14 d、28 d时观察各组血流动力学变化,测定全心重／体重比(whole heart weight/body weight, HW/BW)和左心室重／体重比(left ventricular weight/body weight, LVW/BW),双向电泳-质谱分析技术检测术后28 d心肌组织蛋白质表达谱的变化,RT-PCR技术检测左心室心肌组织ER应激分子GRP78、CRT和CHOP等mRNA表达变化,Western blot分析α-肌动蛋白(α-actin)、GRP78. CRT、CHOP,以及凋亡相关蛋白Bax和Bcl-2等表达变化。
结果发现腹主动脉狭窄诱导大鼠心肌肥大,与假手术组比较,术后7d模型组大鼠血压升高,心功能代偿性增加,HW/BW和LVW/BW显著增加。狭窄术后28 d心肌肥大标志性蛋白肌球蛋白轻链表达上调,而心脏a-actin前体和α-肌球蛋白重链表达下调,Western blot证实模型组心肌组织a-actin表达较假手术组显著增加。其次发现模型组CRT mRNA表达于术后1d即发生显著上调,较假手术组增加136%(P<0.01)；GRP78表达于术后7d显著增加,其高表达均持续至实验结束。长期ER应激触发CHOP凋亡途径,模型组大鼠心肌组织CHOP和促凋亡蛋白Bax表达均于术后14d显著增加,而抗凋亡蛋白Bcl-2表达降低。上述结果提示腹主动脉狭窄早期即可触发ER应激,诱导ER分子伴侣表达增加,ER应激反应可能参与了腹主动脉狭窄致大鼠高血压、心肌肥大过程。CHOP介导的ER应激相关凋亡途径可能参与了心肌肥大及失代偿的调节,决定肥大心肌失代偿的进程。2 ER应激诱导剂TG和TM诱导原代培养心肌细胞肥大和显著ER应激
上述实验发现ER应激参与了腹主动脉狭窄致大鼠心肌肥大的发生、发展过程,为证实ER应激独立诱导心肌肥大,本部分实验在原代培养乳鼠心肌细胞模型上,采用不同浓度TG(1、2.5、5、10、50、70、100 nmol／L)处理原代培养心肌细胞48 h或50 nmol／LTG分别处理心肌细胞12、24、36、48、60、72 h；同时采用不同浓度TM(1、10、100 ng／m1)分别处理心肌细胞48、72、96 h；10-7mmol／L血管紧张素(angiotensin, Ang)Ⅱ处理心肌细胞48 h作为阳性对照。采用培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性和细胞凋亡率检测反映心肌细胞损伤变化,RT-PCR技术观察心肌细胞肥大标志性基因心房钠尿肽(atrial natriuretic peptide, ANP)和脑钠肽(brain natriuretic peptide, BNP) mRNA表达,3H-亮氨酸([3H]-Leucine)掺入技术检测心肌细胞蛋白质合成速率,F-actin染色技术观测心肌细胞骨架改变,同时分析心肌细胞表面积变化。此外采用RT-PCR技术观测ER应激分子CRT、GRP78、PERK、ATF4和CHOP mRNA表达,Western blot技术观测CRT、GRP78、CHOP、Bax和Bel-2蛋白水平改变,同时采用ER特异性荧光染料Dapoxyl观察ER形态变化,免疫荧光技术观察CRT荧光改变。
结果发现,ER应激诱导剂TG和TM以时间和剂量依赖性方式诱导心肌细胞损伤,培养基LDH活性和细胞凋亡率显著增加；同时心肌细胞显著肥大,表现为TG和TM以时间和剂量依赖性方式诱导ANP和BNP mRNA表达、蛋白合成速率和细胞表面积增加；F-actin染色表明TG和TM诱导心肌细胞骨架荧光强度显著增强、应力纤维增加。而且发现50 nmol／L TG作用48 h和10 ng／ml TM作用72 h心肌细胞显著肥大,细胞损伤较轻,是诱导心肌细胞肥大的较适条件。此外,TG以剂量和时间依赖性方式诱导培养心肌细胞显著ER应激,ER应激分子CRT、GRP78表达显著增加；值得注意的是PERK和ATF4 mRNA表达于TG作用24-48 h显著增加,而在60-72 h时显著降低。严重ER应激触发细胞凋亡途径,TG以剂量和时间依赖性方式促进ER凋亡蛋白CHOP表达增加和Bcl-2/Bax比值显著降低。心肌细胞ER染色显示ER形态显著扩张,荧光颗粒分布不均、浓集并出现空泡。免疫荧光显示TG作用后CRT荧光强度增强且向核周浓集。10 ng／ml TM处理72 h心肌细胞也证实上述ER应激改变。上述结果提示ER应激诱导剂TG和TM诱导培养心肌细胞显著肥大和ER应激,而且CHOP凋亡途径激活参与了ER应激诱导的心肌细胞肥大。3 CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程
ER是细胞内重要的Ca2+处理器,细胞内Ca2+升高是触发心肌细胞肥大的基本信号,而CaN可直接受细胞内Ca2+调控,本部分实验旨在探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。不同浓度TG(1、2.5、5、10、50、70、100 nrnol／L)处理原代培养心肌细胞48 h或50 nmol／L TG分别处理心肌细胞12、24、36、48、60、72 h,同时采用10ng／ml TM处理心肌细胞72 h。其次为观察CaN活性抑制后心肌细胞肥大和ER应激变化,在50 nmol／L TG作用48 h诱导心肌细胞肥大模型上,采用CaN活性抑制剂环孢素A(CsA,5μmol/L)预处理心肌细胞10 min后继而培养基内加入50nmol/LTG作用48 h。采用Fluo-3AM染色检测心肌细胞内游离Ca2+水平,超微量Ca2+-ATP酶活性测试盒检测肌浆网／内质网钙-ATPase(sarco／ER Ca2+-ATPase,SERCA)活性,同时采用底物发色法检测心肌细胞CaN活性,采用细胞凋亡率和培养基LDH活性检测反映CaN活性抑制后细胞损伤情况,采用ANP和BNP mRNA表达、蛋白合成速率和细胞表面积检测评价CaN活性抑制后心肌细胞肥大变化。Western blot技术检测心肌细胞SERCA,受磷蛋白(phospholamban, PLB)、MEF2c、p-MEF2c以及CaN活性抑制后ER应激分子CRT、GRP78、CHOP、Bax和Bcl-2等蛋白表达,免疫荧光技术检测MEF2c荧光改变。
结果发现ER应激诱导剂TG以剂量和时间依赖性方式诱导细胞内游离Ca2+水平升高,同时SERCA活性和表达降低、PLB表达增加；此外心肌细胞CaN活性和MEF2c/p-MEF2c蛋白表达显著增加。TM作用心肌细胞也证实ER应激诱导心肌细胞内游离Ca2+水平增加和SERCA活性降低,同时CaN活性升高。免疫荧光显示正常心肌细胞MEF2c主要分布在细胞浆,TG作用后MEF2c向核内转位。CsA显著抑制TG诱导的心肌细胞CaN活性升高,同时阻断TG诱导的心肌细胞肥大,表现为CsA显著阻断TG诱导的心肌细胞ANP、BNP mRNA表达、蛋白质合成速率和细胞表面积增加。免疫荧光发现TG诱导的心肌细胞MEF-2c表达和核转位均显著被CsA抑制。心肌细胞肥大抑制后细胞损伤显著增加,细胞凋亡率和培养基LDH活性升高。此外发现CaN活性抑制没有阻断TG诱导的CRT、GRP78和CHOP表达增加。上述结果提示SERCA活性降低和PLB表达增加可能参与了心肌细胞内游离Ca2+增加,Ca2+可能通过激活CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程。心肌细胞肥大可能是ER应激损伤的代偿性反应,CaN-MEF2c途径阻断显著抑制ER应激诱导的心肌细胞肥大,导致细胞损伤增加,至少部分是通过CHOP介导的ER应激凋亡途径实现的。
通过上述分析,得出如下实验结论：ER应激不仅参与腹主动脉狭窄致高血压大鼠心肌肥大的发生、发展过程,而且独立诱导培养乳鼠心肌细胞肥大发生,CaN-MEF2c信号途径参与了ER应激诱导的心肌细胞肥大发生、发展过程,CaN活性抑制显著阻断ER应激诱导的心肌细胞肥大,同时细胞损伤显著增加。提示ER应激是心肌肥大的发病学因素之一,为心肌肥大、心力衰竭的临床治疗提供了新的靶点。
Cardiac hypertrophy is an adaptive response triggered by many physiological and pathological conditions. However, epidemiological data indicate that chronic cardiac hypertrophy takes part in the development of various cardiovascular diseases such as dilated cardiomyopathy, heart failure and sudden death, which severely endanger human lives. So it is very important to interpret the pathogenesis of hypertrophy for clinical prevention and cure of hypertrophy and heart failure.
Endoplasmic reticulum (ER) is an organelle regulating intracellular Ca2+, folding of secreted and membrane protein and cell apoptosis. Various stimuli, such as ER-Ca2+depletion, elevated protein synthesis, ischemia and hypoxia, disturb ER homeostasis and result in ER stress. In response to ER stress, ER chaperones such as calreticulin (CRT) and glucose-regulated proteins (GRPs) are upregulated to enhance the ER ability to regulate intracellular Ca2+level and handle with the unfolded proteins, which is cardioprotective. However, when ER stress is excessive and/or prolonged, the ER-related apoptotic process is initiated by induction of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and caspase-12 activation, which lead to cell apoptosis and tissue injury. Protein kinase R-like ER kinase (PERK) is an important ER transmembrane protein, the relocation of GRP78 from the luminal domain of PERK to misfolded proteins leads to PERK activation, which induces translation of activating transcription factor 4 (ATF4) and its target genes such as CHOP, thus contributing to apoptosis.
Accumulating evidence has demonstrated that apoptosis initiated by ER stress is involved in the pathogenesis of neurodegeneration, diabetic cardiomyopathy, and ischemia/reperfusion injury. Moreover, the expression of CRT, GRP78, and GRP94 increases significantly during cardiac hypertrophy, and CHOP-mediated apoptosis contributes to the development from cardiac hypertrophy to heart failure, which indicates that ER stress is involved in the development of hypertrophy, but the roles of ER stress in the development of hypertrophy is still unclear. Intracellular Ca2+ regulated predominantly by ER is a ubiquitous second messenger whose level is elevated significantly in hypertrophied cardiac myocytes. Calcineurin (CaN) is a highly conserved, Ca2+/calmodulin-dependent serine/threonine phosphatase composed of catalytic subunit A and regulatory subunit B, which has important roles in regulating T-cell activation, hypertrophy, cell cycle and so on. Myocyte enhancer factor 2c (MEF2c) is the first gene of the MEF2 family found to be expressed during cardiac development and a substrate for CaN in myocardium. However, whether this pathway is involved in ER stress-induced cardiac hypertrophy is unknown.
In this present study, we explored the roles of ER stress in the development of cardiac hypertrophy induced by abdominal aortic constriction in rats. Next, ER stress in cultured neonatal rat cardiomyocytes was induced by two ER stress inducers thapsigargin (TG), which depletes Ca2+from ER, and tunicamycin (TM), which inhibits protein N-linked glycosylation. We explored the effects of TG and TM on ER stress and cardiac hypertrophy in cardiomyocytes, and also investigated the roles of CaN-MEF2c signal pathway in the development of cardiac hypertrophy in cardiomyocytes. The methods and results were as follows:
1 ER stress was involved in the development of myocardial hypertrophy induced by abdominal aortic constriction in rats
This part of work aimed to explore the roles of ER stress response in the development of myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and sham group (n=40). The rats in model group were operated on abdominal aortic constriction, while the abdominal aorta in sham group was only separated but not constricted. Hemodynamic changes, whole heart weight-to-body weight ratio. (HW/BW) and left ventricular weight-to-body weight ratio (LVW/BW) were measured at 1 d,3 d,7 d,14 d and 28 d after surgery, respectively.2-D electrophoresis and mass spectrometry were used to identify the proteomic profile in hypertrophic myocardium at 28 d after surgery. The mRNA expression of GRP78, CRT and CHOP, which were important markers of ER stress, were detected by RT-PCR, and western blot was used to assess the protein level of a-actin, GRP78, CRT, CHOP, and apoptosis-associated protein Bax and Bcl-2.
It was found that abdominal aortic constriction induced significant myocardial hypertrophy in rats. Compared with sham group, the blood pressure, LVW/BW, and HW/BW in model group increased significantly and cardiac function enhanced compensatively at 7 d after surgery, which increased progressively during the experiment. The expression of myosin light chain increased andα-actin proprotein andα-myosin heavy chain decreased, all of which are the markers of hypertrophy, at 28 d after abdominal aortic constriction. The upregulation ofα-actin expression in model group was also verified by western blot. In addition, as early as 1 d after surgery, the mRNA level of CRT in model group increased by 136%(P<0.01) compared with sham group. The mRNA and protein expression of GRP78 increased at 7 d after surgery, and the expression of CRT and GRP78 sustained high level till to the end of experiment. Prolonged ER stress triggered myocyte apoptosis pathway, compared with sham group, both the expression of CHOP and Bax in model group increased, while the expression of anti-apoptotic protein Bcl-2 decreased at 14 d after surgery. The results above indicated that abdominal aortic constriction induced significant upregulation in ER molecular chaperones at early stage of post-surgery, ER stress response may take part in the development of cardiac hypertrophy. CHOP-mediated cell apoptosis pathway may be involved in the regulation of development in myocardial hypertrophy and heart failure, and control the progression of decompensative hypertrophy.
2 ER stress inducers TG and TM induced cardiac hypertrophy and ER stress in neonatal rat cardiomyocytes
The results above indicated that ER stress was involved in the development of hypertrophy in rat heart. To verify ER stress could induce hypertrophy independently, primary cultures from neonatal rats were incubated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12, 24,36,48,60, and 72 h, respectively; moreover,1,10,100 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 48,72,96 h, respectively.10"7 mmol/L angiotensin (Ang)Ⅱfor 48 h treatment was as positive control. The detections of lactate dehydrogenase (LDH) activity in medium and cell apoptosis rate were used to reflect cell injury in cardiomyocytes. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which were hypertrophic gene markers, were detected by RT-PCR. Total protein synthesis rate in cardiomyocytes was evaluated by incorporation of 3H-Leucine. F-actin fluorescence staining was employed to view cytoskeleton in cardiomyocytes, and cell surface area in cardiomyocytes was analyzed. In addition, RT-PCR was used to detect the mRNA expression of ER stress molecules, including CRT, GRP78, PERK, ATF4, and CHOP. Western blot was employed to detect the protein expression of ER stress proteins such as CRT, GRP78, CHOP and apoptosis-related Bax and Bcl-2. ER-specific dye Dapoxyl was used to observe ER morph in freshly viable cardiomyocytes, and CRT immunofluorescence in cardiomyocytes was viewed under a confocal laser scanning microscope.
The results were as follows:ER stress inducers TG and TM induced cell injury in a time-and dose-dependent manner in cardiomyocytes, as shown as the increase in LDH activity in the medium and cell apoptosis rate. Meanwhile, cardiac hypertrophy in cardiomyocytes was induced by TG and TM in a time-and dose-dependent manner, as characterized by the elevation in the ANP and BNP mRNA expression, in the protein synthesis rate and in the cell surface area. F-actin staining observed that the F-actin fluorescence intensity was augmented significantly in TG-and TM-treated cardiomyocytes. It was found that 50 nmol/L TG for 48 h or 10 ng/ml TM for 72 h treatment was the suitable condition to induce cardiac hypertrophy without severe injury in cardiomyocytes. In addition, it was found that TG induced significant ER stress response in a time-and dose-dependent manner in cardiomyocytes, as characterized by the increase in CRT and GRP78 expression. Of interest, the mRNA expression of PERK and ATF4 in TG-treated cardiomyocytes increased at 24-48 h, and decreased at 60-72 h. Severe or prolonged ER stress triggered CHOP-mediated apoptosis pathway, TG upregulated CHOP expression and downregulated Bcl-2 to Bax ratio in a time-and dose-dependent manner in cardiomyocytes. ER staining in freshly viable cardiomyocytes found that the structure of the ER in TG-induced cardiomyocytes severely destroyed and vacuole appeared. The ER stress profile showed above was confirmed in cardiomyocytes treated with 10 ng/ml TM for 72 h. These results indicated that ER stress inducers induced significant hypertrophy in parallel with ER stress in cardiomyocytes, and CHOP-mediated apoptosis pathway was involved in the development of cardiac hypertrophy in cardiomyocytes.
3 CaN-MEF2c signal pathway was involved in ER stress-induced cardiac hypertrophy in cardiomyocytes
Cytoplasmic Ca2+, which regulates CaN directly, is elevated significantly and plays an essential role in triggering the cardiac hypertrophy response. This part of work was to investigate the roles of CaN-MEF2c pathway in the development of cardiac hypertrophy induced by ER stress in cardiomyocytes. Cardiomyocytes was treated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12,24,36,48,60, and 72 h, respectively; moreover,10 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 72 h. In addition, to explore the effects of CaN activity inhibition on ER stress-induced cardiac hypertrophy and CaN-MEF2c pathway, cardiomyocytes were pretreated with 5μmol/L cyclosporine A (CsA) for 10 min before 50 nmol/L TG for 48 h treatment, which induced notable cardiac hypertrophy. Fluo-3 AM staining was used to detect the relative Ca2+concentration in cytoplasm. Sarco/ER Ca2+-ATPase (SERCA) activity was measured by a Ca2+-ATPase assay kit according to the manufacturer's instructions, and CaN activity was measured by use of p-nitrophenyl phonphate. Analysis of LDH activity in the medium and cell apoptosis rate was used to evaluate cell injury in TG induced-cardiomyocytes with CsA pretreatment. Cardiac hypertrophy in TG induced-cardiomyocytes with CsA pretreatment was evaluated by analysis of the mRNA expression of ANP and BNP, the protein synthesis rate and the cell surface area. Western blot was used to detect the protein expression of SERCA, phospholamban (PLB), MEF2c, p-MEF2c and ER stress-related proteins such as CRT, GRP78, CHOP, Bax and Bcl-2. Immunofluorescence was performed to observe the change of MEF2c fluorescence in cardiomyocytes.
The results were as follows:TG induced significant elevation of intracellular free Ca2+level, CaN activity, and MEF2c/p-MEF2c expression in a dose-and time dependent manner in cardiomyocytes; meanwhile, the SERCA activity decreased in TG-treated cardiomyocytes. The elevation of intracellular Ca+level and CaN activity as well as the decrease of SERCA activity were also verified in TM-treated cardiomyocytes. Immunofluorescence showed that MEF2c localized predominately in cytoplasm in control cardiomyocytes, while MEF2c transferred to nuclei in cardiomyocytes after TG treatment. CaN activity in TG-treated cardiomyocytes was blocked significantly by CsA pretreatment. The cardiac hypertrophy was prevented by CaN activity inhibition with CsA, as shown as the decrease in ANP and BNP mRNA expression, protein synthesis rate and cell surface area. After the inhibition of CaN activity, the MEF2c nuclear translocation was also prevented in TG-treated cardiomyocytes. Meanwhile, the cell injury increased in TG-treated cardiomyocytes with CsA pretreatment, the LDH activity in the medium and cell apoptosis rate increased significantly compared with control cardiomyocytes. In addition, the upregulation of CRT, GRP78 and CHOP in TG-treated cardiomyocytes was not blocked by CsA pretreatment. These results above indicated that the decrease in SERCA activity and the increase of PLB expression may take part in the elevation of intracellular free Ca2+, CaN-MEF2c signal pathway activated by Ca2+was involved in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes. Cardiac hypertrophy was a compensatory response to ER stress-induced injury, the prevention of CaN-MEF2c pathway inhibited cardiac hypertrophy induced by ER stress in cardiomyocytes and resulted in the increase of cell injury, at least in part through an ER stress-mediated cell apoptotic pathway.
The conclusions were as followes:ER stress is not only involved in the deyelopment of myocardial hypertrophy induced by abdominal aortic constriction in rats, but also induces cardiac hypertrophy independently in cultured neonatal cardiomyocytes. CaN-MEF2c pathway takes part in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes, and CaN activity inhibition leads to the prevention of cardiac hypertrophy and the increase of cell injury. Our findings suggest that ER stress induces cardiac hypertrophy independently and may be one of pathogenic factors of cardiac hypertrophy, thus providing new insight into the development of novel therapeutic strategies for cardiac hypertrophy.
内质网(endoplasmic reticulum, ER)是调节细胞内钙稳态和膜型／分泌型蛋白质合成、折叠的重要细胞器,对维持心肌细胞钙和功能蛋白质稳态具有重要作用。ER对应激刺激非常敏感,多种因素如钙紊乱、蛋白合成增加、缺血、缺氧等因素均可触发ER应激。适当的ER应激诱导ER分子伴侣如葡萄糖调节蛋白(glucose-regulated proteins, GRPs)、钙网蛋白(calreticulin, CRT)等表达上调,增强ER钙调节和处理未折叠蛋白的能力,具有细胞保护作用；但是,持续或严重的ER应激可触发ER凋亡信号通路,促进ER重要的促凋亡蛋白C/EBP homologous protein (CHOP)表达增加和caspase-12剪切活化,导致细胞凋亡和组织损伤。蛋白激酶R样ER激酶(protein kinase R-like ER kinase, PERK)是重要的ER跨膜蛋白,ER应激时GRP78与PERK的解离激活PERK,诱导激活型转录因子4 (activating transcription factor 4, ATF4)转录及其下游靶分子CHOP表达,促进细胞凋亡。
多种证据表明ER应激与神经退行性变、糖尿病性心肌病和缺血／再灌注损伤等多种疾病密切相关。研究发现心肌肥大时ER应激分子CRT、GRP78、GRP94等表达显著上调,CHOP介导的ER应激凋亡通路参与了心力衰竭过程中的细胞凋亡,提示心肌肥大过程中存在ER应激反应,但ER应激在心肌肥大发生、发展中的变化及作用目前并不清楚。心肌细胞内Ca2+水平升高参与了心肌肥大的发生,是触发心肌肥大反应的基本信号。钙调神经磷酸酶(calcineurin, CaN)是一种受细胞浆Ca2+及钙调素(calmodulin, CaM)调控的蛋白丝氨酸／苏氨酸磷酸酶,由催化亚基CnA和调节亚基CnB组成,在T细胞活化、心肌肥大、细胞周期调控等多种生物学过程中发挥关键性的调控作用。心肌细胞增强因子2c (myocyte enhancer factor 2c, MEF2c)是MEF2c家族中发现的第一个参与心脏发育的分子,并被CaN激活,但是CaN-MEF2c信号途径在ER应激诱导心肌肥大中的作用目前尚未报道。
本工作首先在大鼠腹主动脉狭窄致高血压、心肌肥大模型上,探讨ER应激在心肌肥大发生、发展过程中的作用；其次采用ER应激诱导剂毒胡萝卜素(thapsigargin, TG,抑制ER钙泵继而排空ER内Ca2+)和衣霉素(tunicamycin, TM,抑制ER内蛋白质N-端糖基化)作用于原代培养乳鼠心肌细胞,观察TG和TM对心肌细胞肥大和ER应激的影响,并探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。主要实验方法和结果如下：
1 ER应激参与大鼠腹主动脉狭窄致高血压心肌肥大发生、发展过程
本部分实验旨在观察ER应激反应在腹主动脉狭窄致高血压大鼠心肌肥大发生、发展过程中的作用。健康雄性Wistar大鼠85只,随机分为模型组(n=45)和假手术组(n=40),模型组行腹主动脉狭窄术,假手术组仅分离腹主动脉不行狭窄术,分别于术后1 d、3 d、7 d、14 d、28 d时观察各组血流动力学变化,测定全心重／体重比(whole heart weight/body weight, HW/BW)和左心室重／体重比(left ventricular weight/body weight, LVW/BW),双向电泳-质谱分析技术检测术后28 d心肌组织蛋白质表达谱的变化,RT-PCR技术检测左心室心肌组织ER应激分子GRP78、CRT和CHOP等mRNA表达变化,Western blot分析α-肌动蛋白(α-actin)、GRP78. CRT、CHOP,以及凋亡相关蛋白Bax和Bcl-2等表达变化。
结果发现腹主动脉狭窄诱导大鼠心肌肥大,与假手术组比较,术后7d模型组大鼠血压升高,心功能代偿性增加,HW/BW和LVW/BW显著增加。狭窄术后28 d心肌肥大标志性蛋白肌球蛋白轻链表达上调,而心脏a-actin前体和α-肌球蛋白重链表达下调,Western blot证实模型组心肌组织a-actin表达较假手术组显著增加。其次发现模型组CRT mRNA表达于术后1d即发生显著上调,较假手术组增加136%(P<0.01)；GRP78表达于术后7d显著增加,其高表达均持续至实验结束。长期ER应激触发CHOP凋亡途径,模型组大鼠心肌组织CHOP和促凋亡蛋白Bax表达均于术后14d显著增加,而抗凋亡蛋白Bcl-2表达降低。上述结果提示腹主动脉狭窄早期即可触发ER应激,诱导ER分子伴侣表达增加,ER应激反应可能参与了腹主动脉狭窄致大鼠高血压、心肌肥大过程。CHOP介导的ER应激相关凋亡途径可能参与了心肌肥大及失代偿的调节,决定肥大心肌失代偿的进程。2 ER应激诱导剂TG和TM诱导原代培养心肌细胞肥大和显著ER应激
上述实验发现ER应激参与了腹主动脉狭窄致大鼠心肌肥大的发生、发展过程,为证实ER应激独立诱导心肌肥大,本部分实验在原代培养乳鼠心肌细胞模型上,采用不同浓度TG(1、2.5、5、10、50、70、100 nmol／L)处理原代培养心肌细胞48 h或50 nmol／LTG分别处理心肌细胞12、24、36、48、60、72 h；同时采用不同浓度TM(1、10、100 ng／m1)分别处理心肌细胞48、72、96 h；10-7mmol／L血管紧张素(angiotensin, Ang)Ⅱ处理心肌细胞48 h作为阳性对照。采用培养基乳酸脱氢酶(lactate dehydrogenase, LDH)活性和细胞凋亡率检测反映心肌细胞损伤变化,RT-PCR技术观察心肌细胞肥大标志性基因心房钠尿肽(atrial natriuretic peptide, ANP)和脑钠肽(brain natriuretic peptide, BNP) mRNA表达,3H-亮氨酸([3H]-Leucine)掺入技术检测心肌细胞蛋白质合成速率,F-actin染色技术观测心肌细胞骨架改变,同时分析心肌细胞表面积变化。此外采用RT-PCR技术观测ER应激分子CRT、GRP78、PERK、ATF4和CHOP mRNA表达,Western blot技术观测CRT、GRP78、CHOP、Bax和Bel-2蛋白水平改变,同时采用ER特异性荧光染料Dapoxyl观察ER形态变化,免疫荧光技术观察CRT荧光改变。
结果发现,ER应激诱导剂TG和TM以时间和剂量依赖性方式诱导心肌细胞损伤,培养基LDH活性和细胞凋亡率显著增加；同时心肌细胞显著肥大,表现为TG和TM以时间和剂量依赖性方式诱导ANP和BNP mRNA表达、蛋白合成速率和细胞表面积增加；F-actin染色表明TG和TM诱导心肌细胞骨架荧光强度显著增强、应力纤维增加。而且发现50 nmol／L TG作用48 h和10 ng／ml TM作用72 h心肌细胞显著肥大,细胞损伤较轻,是诱导心肌细胞肥大的较适条件。此外,TG以剂量和时间依赖性方式诱导培养心肌细胞显著ER应激,ER应激分子CRT、GRP78表达显著增加；值得注意的是PERK和ATF4 mRNA表达于TG作用24-48 h显著增加,而在60-72 h时显著降低。严重ER应激触发细胞凋亡途径,TG以剂量和时间依赖性方式促进ER凋亡蛋白CHOP表达增加和Bcl-2/Bax比值显著降低。心肌细胞ER染色显示ER形态显著扩张,荧光颗粒分布不均、浓集并出现空泡。免疫荧光显示TG作用后CRT荧光强度增强且向核周浓集。10 ng／ml TM处理72 h心肌细胞也证实上述ER应激改变。上述结果提示ER应激诱导剂TG和TM诱导培养心肌细胞显著肥大和ER应激,而且CHOP凋亡途径激活参与了ER应激诱导的心肌细胞肥大。3 CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程
ER是细胞内重要的Ca2+处理器,细胞内Ca2+升高是触发心肌细胞肥大的基本信号,而CaN可直接受细胞内Ca2+调控,本部分实验旨在探讨CaN-MEF2c信号途径在ER应激诱导心肌细胞肥大过程中的作用。不同浓度TG(1、2.5、5、10、50、70、100 nrnol／L)处理原代培养心肌细胞48 h或50 nmol／L TG分别处理心肌细胞12、24、36、48、60、72 h,同时采用10ng／ml TM处理心肌细胞72 h。其次为观察CaN活性抑制后心肌细胞肥大和ER应激变化,在50 nmol／L TG作用48 h诱导心肌细胞肥大模型上,采用CaN活性抑制剂环孢素A(CsA,5μmol/L)预处理心肌细胞10 min后继而培养基内加入50nmol/LTG作用48 h。采用Fluo-3AM染色检测心肌细胞内游离Ca2+水平,超微量Ca2+-ATP酶活性测试盒检测肌浆网／内质网钙-ATPase(sarco／ER Ca2+-ATPase,SERCA)活性,同时采用底物发色法检测心肌细胞CaN活性,采用细胞凋亡率和培养基LDH活性检测反映CaN活性抑制后细胞损伤情况,采用ANP和BNP mRNA表达、蛋白合成速率和细胞表面积检测评价CaN活性抑制后心肌细胞肥大变化。Western blot技术检测心肌细胞SERCA,受磷蛋白(phospholamban, PLB)、MEF2c、p-MEF2c以及CaN活性抑制后ER应激分子CRT、GRP78、CHOP、Bax和Bcl-2等蛋白表达,免疫荧光技术检测MEF2c荧光改变。
结果发现ER应激诱导剂TG以剂量和时间依赖性方式诱导细胞内游离Ca2+水平升高,同时SERCA活性和表达降低、PLB表达增加；此外心肌细胞CaN活性和MEF2c/p-MEF2c蛋白表达显著增加。TM作用心肌细胞也证实ER应激诱导心肌细胞内游离Ca2+水平增加和SERCA活性降低,同时CaN活性升高。免疫荧光显示正常心肌细胞MEF2c主要分布在细胞浆,TG作用后MEF2c向核内转位。CsA显著抑制TG诱导的心肌细胞CaN活性升高,同时阻断TG诱导的心肌细胞肥大,表现为CsA显著阻断TG诱导的心肌细胞ANP、BNP mRNA表达、蛋白质合成速率和细胞表面积增加。免疫荧光发现TG诱导的心肌细胞MEF-2c表达和核转位均显著被CsA抑制。心肌细胞肥大抑制后细胞损伤显著增加,细胞凋亡率和培养基LDH活性升高。此外发现CaN活性抑制没有阻断TG诱导的CRT、GRP78和CHOP表达增加。上述结果提示SERCA活性降低和PLB表达增加可能参与了心肌细胞内游离Ca2+增加,Ca2+可能通过激活CaN-MEF2c信号途径参与ER应激诱导的心肌细胞肥大过程。心肌细胞肥大可能是ER应激损伤的代偿性反应,CaN-MEF2c途径阻断显著抑制ER应激诱导的心肌细胞肥大,导致细胞损伤增加,至少部分是通过CHOP介导的ER应激凋亡途径实现的。
通过上述分析,得出如下实验结论：ER应激不仅参与腹主动脉狭窄致高血压大鼠心肌肥大的发生、发展过程,而且独立诱导培养乳鼠心肌细胞肥大发生,CaN-MEF2c信号途径参与了ER应激诱导的心肌细胞肥大发生、发展过程,CaN活性抑制显著阻断ER应激诱导的心肌细胞肥大,同时细胞损伤显著增加。提示ER应激是心肌肥大的发病学因素之一,为心肌肥大、心力衰竭的临床治疗提供了新的靶点。
Cardiac hypertrophy is an adaptive response triggered by many physiological and pathological conditions. However, epidemiological data indicate that chronic cardiac hypertrophy takes part in the development of various cardiovascular diseases such as dilated cardiomyopathy, heart failure and sudden death, which severely endanger human lives. So it is very important to interpret the pathogenesis of hypertrophy for clinical prevention and cure of hypertrophy and heart failure.
Endoplasmic reticulum (ER) is an organelle regulating intracellular Ca2+, folding of secreted and membrane protein and cell apoptosis. Various stimuli, such as ER-Ca2+depletion, elevated protein synthesis, ischemia and hypoxia, disturb ER homeostasis and result in ER stress. In response to ER stress, ER chaperones such as calreticulin (CRT) and glucose-regulated proteins (GRPs) are upregulated to enhance the ER ability to regulate intracellular Ca2+level and handle with the unfolded proteins, which is cardioprotective. However, when ER stress is excessive and/or prolonged, the ER-related apoptotic process is initiated by induction of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) and caspase-12 activation, which lead to cell apoptosis and tissue injury. Protein kinase R-like ER kinase (PERK) is an important ER transmembrane protein, the relocation of GRP78 from the luminal domain of PERK to misfolded proteins leads to PERK activation, which induces translation of activating transcription factor 4 (ATF4) and its target genes such as CHOP, thus contributing to apoptosis.
Accumulating evidence has demonstrated that apoptosis initiated by ER stress is involved in the pathogenesis of neurodegeneration, diabetic cardiomyopathy, and ischemia/reperfusion injury. Moreover, the expression of CRT, GRP78, and GRP94 increases significantly during cardiac hypertrophy, and CHOP-mediated apoptosis contributes to the development from cardiac hypertrophy to heart failure, which indicates that ER stress is involved in the development of hypertrophy, but the roles of ER stress in the development of hypertrophy is still unclear. Intracellular Ca2+ regulated predominantly by ER is a ubiquitous second messenger whose level is elevated significantly in hypertrophied cardiac myocytes. Calcineurin (CaN) is a highly conserved, Ca2+/calmodulin-dependent serine/threonine phosphatase composed of catalytic subunit A and regulatory subunit B, which has important roles in regulating T-cell activation, hypertrophy, cell cycle and so on. Myocyte enhancer factor 2c (MEF2c) is the first gene of the MEF2 family found to be expressed during cardiac development and a substrate for CaN in myocardium. However, whether this pathway is involved in ER stress-induced cardiac hypertrophy is unknown.
In this present study, we explored the roles of ER stress in the development of cardiac hypertrophy induced by abdominal aortic constriction in rats. Next, ER stress in cultured neonatal rat cardiomyocytes was induced by two ER stress inducers thapsigargin (TG), which depletes Ca2+from ER, and tunicamycin (TM), which inhibits protein N-linked glycosylation. We explored the effects of TG and TM on ER stress and cardiac hypertrophy in cardiomyocytes, and also investigated the roles of CaN-MEF2c signal pathway in the development of cardiac hypertrophy in cardiomyocytes. The methods and results were as follows:
1 ER stress was involved in the development of myocardial hypertrophy induced by abdominal aortic constriction in rats
This part of work aimed to explore the roles of ER stress response in the development of myocardial hypertrophy induced by abdominal aortic constriction in rats. Healthy male Wistar rats were randomly divided into model group (n=45) and sham group (n=40). The rats in model group were operated on abdominal aortic constriction, while the abdominal aorta in sham group was only separated but not constricted. Hemodynamic changes, whole heart weight-to-body weight ratio. (HW/BW) and left ventricular weight-to-body weight ratio (LVW/BW) were measured at 1 d,3 d,7 d,14 d and 28 d after surgery, respectively.2-D electrophoresis and mass spectrometry were used to identify the proteomic profile in hypertrophic myocardium at 28 d after surgery. The mRNA expression of GRP78, CRT and CHOP, which were important markers of ER stress, were detected by RT-PCR, and western blot was used to assess the protein level of a-actin, GRP78, CRT, CHOP, and apoptosis-associated protein Bax and Bcl-2.
It was found that abdominal aortic constriction induced significant myocardial hypertrophy in rats. Compared with sham group, the blood pressure, LVW/BW, and HW/BW in model group increased significantly and cardiac function enhanced compensatively at 7 d after surgery, which increased progressively during the experiment. The expression of myosin light chain increased andα-actin proprotein andα-myosin heavy chain decreased, all of which are the markers of hypertrophy, at 28 d after abdominal aortic constriction. The upregulation ofα-actin expression in model group was also verified by western blot. In addition, as early as 1 d after surgery, the mRNA level of CRT in model group increased by 136%(P<0.01) compared with sham group. The mRNA and protein expression of GRP78 increased at 7 d after surgery, and the expression of CRT and GRP78 sustained high level till to the end of experiment. Prolonged ER stress triggered myocyte apoptosis pathway, compared with sham group, both the expression of CHOP and Bax in model group increased, while the expression of anti-apoptotic protein Bcl-2 decreased at 14 d after surgery. The results above indicated that abdominal aortic constriction induced significant upregulation in ER molecular chaperones at early stage of post-surgery, ER stress response may take part in the development of cardiac hypertrophy. CHOP-mediated cell apoptosis pathway may be involved in the regulation of development in myocardial hypertrophy and heart failure, and control the progression of decompensative hypertrophy.
2 ER stress inducers TG and TM induced cardiac hypertrophy and ER stress in neonatal rat cardiomyocytes
The results above indicated that ER stress was involved in the development of hypertrophy in rat heart. To verify ER stress could induce hypertrophy independently, primary cultures from neonatal rats were incubated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12, 24,36,48,60, and 72 h, respectively; moreover,1,10,100 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 48,72,96 h, respectively.10"7 mmol/L angiotensin (Ang)Ⅱfor 48 h treatment was as positive control. The detections of lactate dehydrogenase (LDH) activity in medium and cell apoptosis rate were used to reflect cell injury in cardiomyocytes. The mRNA expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which were hypertrophic gene markers, were detected by RT-PCR. Total protein synthesis rate in cardiomyocytes was evaluated by incorporation of 3H-Leucine. F-actin fluorescence staining was employed to view cytoskeleton in cardiomyocytes, and cell surface area in cardiomyocytes was analyzed. In addition, RT-PCR was used to detect the mRNA expression of ER stress molecules, including CRT, GRP78, PERK, ATF4, and CHOP. Western blot was employed to detect the protein expression of ER stress proteins such as CRT, GRP78, CHOP and apoptosis-related Bax and Bcl-2. ER-specific dye Dapoxyl was used to observe ER morph in freshly viable cardiomyocytes, and CRT immunofluorescence in cardiomyocytes was viewed under a confocal laser scanning microscope.
The results were as follows:ER stress inducers TG and TM induced cell injury in a time-and dose-dependent manner in cardiomyocytes, as shown as the increase in LDH activity in the medium and cell apoptosis rate. Meanwhile, cardiac hypertrophy in cardiomyocytes was induced by TG and TM in a time-and dose-dependent manner, as characterized by the elevation in the ANP and BNP mRNA expression, in the protein synthesis rate and in the cell surface area. F-actin staining observed that the F-actin fluorescence intensity was augmented significantly in TG-and TM-treated cardiomyocytes. It was found that 50 nmol/L TG for 48 h or 10 ng/ml TM for 72 h treatment was the suitable condition to induce cardiac hypertrophy without severe injury in cardiomyocytes. In addition, it was found that TG induced significant ER stress response in a time-and dose-dependent manner in cardiomyocytes, as characterized by the increase in CRT and GRP78 expression. Of interest, the mRNA expression of PERK and ATF4 in TG-treated cardiomyocytes increased at 24-48 h, and decreased at 60-72 h. Severe or prolonged ER stress triggered CHOP-mediated apoptosis pathway, TG upregulated CHOP expression and downregulated Bcl-2 to Bax ratio in a time-and dose-dependent manner in cardiomyocytes. ER staining in freshly viable cardiomyocytes found that the structure of the ER in TG-induced cardiomyocytes severely destroyed and vacuole appeared. The ER stress profile showed above was confirmed in cardiomyocytes treated with 10 ng/ml TM for 72 h. These results indicated that ER stress inducers induced significant hypertrophy in parallel with ER stress in cardiomyocytes, and CHOP-mediated apoptosis pathway was involved in the development of cardiac hypertrophy in cardiomyocytes.
3 CaN-MEF2c signal pathway was involved in ER stress-induced cardiac hypertrophy in cardiomyocytes
Cytoplasmic Ca2+, which regulates CaN directly, is elevated significantly and plays an essential role in triggering the cardiac hypertrophy response. This part of work was to investigate the roles of CaN-MEF2c pathway in the development of cardiac hypertrophy induced by ER stress in cardiomyocytes. Cardiomyocytes was treated with the indicated concentration of TG (1,2.5,5,10,20,50,70,100 nmol/L) for 48 h, or with 50 nmol/L TG for 12,24,36,48,60, and 72 h, respectively; moreover,10 ng/ml TM, another ER stress inductor, was used to treat cardiomyocytes for 72 h. In addition, to explore the effects of CaN activity inhibition on ER stress-induced cardiac hypertrophy and CaN-MEF2c pathway, cardiomyocytes were pretreated with 5μmol/L cyclosporine A (CsA) for 10 min before 50 nmol/L TG for 48 h treatment, which induced notable cardiac hypertrophy. Fluo-3 AM staining was used to detect the relative Ca2+concentration in cytoplasm. Sarco/ER Ca2+-ATPase (SERCA) activity was measured by a Ca2+-ATPase assay kit according to the manufacturer's instructions, and CaN activity was measured by use of p-nitrophenyl phonphate. Analysis of LDH activity in the medium and cell apoptosis rate was used to evaluate cell injury in TG induced-cardiomyocytes with CsA pretreatment. Cardiac hypertrophy in TG induced-cardiomyocytes with CsA pretreatment was evaluated by analysis of the mRNA expression of ANP and BNP, the protein synthesis rate and the cell surface area. Western blot was used to detect the protein expression of SERCA, phospholamban (PLB), MEF2c, p-MEF2c and ER stress-related proteins such as CRT, GRP78, CHOP, Bax and Bcl-2. Immunofluorescence was performed to observe the change of MEF2c fluorescence in cardiomyocytes.
The results were as follows:TG induced significant elevation of intracellular free Ca2+level, CaN activity, and MEF2c/p-MEF2c expression in a dose-and time dependent manner in cardiomyocytes; meanwhile, the SERCA activity decreased in TG-treated cardiomyocytes. The elevation of intracellular Ca+level and CaN activity as well as the decrease of SERCA activity were also verified in TM-treated cardiomyocytes. Immunofluorescence showed that MEF2c localized predominately in cytoplasm in control cardiomyocytes, while MEF2c transferred to nuclei in cardiomyocytes after TG treatment. CaN activity in TG-treated cardiomyocytes was blocked significantly by CsA pretreatment. The cardiac hypertrophy was prevented by CaN activity inhibition with CsA, as shown as the decrease in ANP and BNP mRNA expression, protein synthesis rate and cell surface area. After the inhibition of CaN activity, the MEF2c nuclear translocation was also prevented in TG-treated cardiomyocytes. Meanwhile, the cell injury increased in TG-treated cardiomyocytes with CsA pretreatment, the LDH activity in the medium and cell apoptosis rate increased significantly compared with control cardiomyocytes. In addition, the upregulation of CRT, GRP78 and CHOP in TG-treated cardiomyocytes was not blocked by CsA pretreatment. These results above indicated that the decrease in SERCA activity and the increase of PLB expression may take part in the elevation of intracellular free Ca2+, CaN-MEF2c signal pathway activated by Ca2+was involved in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes. Cardiac hypertrophy was a compensatory response to ER stress-induced injury, the prevention of CaN-MEF2c pathway inhibited cardiac hypertrophy induced by ER stress in cardiomyocytes and resulted in the increase of cell injury, at least in part through an ER stress-mediated cell apoptotic pathway.
The conclusions were as followes:ER stress is not only involved in the deyelopment of myocardial hypertrophy induced by abdominal aortic constriction in rats, but also induces cardiac hypertrophy independently in cultured neonatal cardiomyocytes. CaN-MEF2c pathway takes part in the development of ER stress-induced cardiac hypertrophy in cardiomyocytes, and CaN activity inhibition leads to the prevention of cardiac hypertrophy and the increase of cell injury. Our findings suggest that ER stress induces cardiac hypertrophy independently and may be one of pathogenic factors of cardiac hypertrophy, thus providing new insight into the development of novel therapeutic strategies for cardiac hypertrophy.
引文
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23 Sakuma K, Akiho M, Nakashima H, Nakao R, Hirata M, Inashima S, Yamaguchi A, Yasuhara M. Cyclosporin A modulates cellular localization of MEF2C protein and blocks fiber hypertrophy in the overloaded soleus muscle of mice. Acta Neuropathol.2008,115:663-674.
2 Kaufman RJ, Stress signaling from the lumen of the endoplasmic reticulum: coordination of gene transcription and translational controls. Gene Dev.1999,13: 1211-1233.
3 Ron D. Translational control in the endoplasmic reticulum stress response. J Clin Invest.2002,110:1383-1388.
4 Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ.2004,11:381-389.
5 Zinszner H, Kuroda M, Wang X, Batchvarova N, Lightfoot RT, Remotti H, Stevens JL, Ron D. CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum. Genes Dev.1998,12:982-995.
6 Friedman AD. GADD153/CHOP, a DNA damage-inducible protein, reduced CAAT/enhancer binding protein activities and increased apoptosis in 32D c13 myeloid cells. Cancer Res.1996,56:3250-3256.
7 McCullough KD, Martindale JL, Klotz LO, Aw TY, Holbrook NJ. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol Cell Biol.2001,21:1249-1259.
8 Nieto-Miguel T, Fonteriz RI, Vay L, Gajate C, Lopez-Hernandez S, Mollinedo F. Endoplasmic reticulum stress in the proapoptotic action of edelfosine in solid tumor cells. Cancer Res.2007,67:10368-10378.
9 Gotoh T, Terada K, Oyadomari S, Mori M. hsp70-DnaJ chaperone pair prevents nitric oxide-and CHOP-induced apoptosis by inhibiting translocation of Bax to mitochondria. Cell Death Differ.2004,11:390-402.
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15 Frey N, McKinsey TA, Olson EN. Decoding calcium signals involved in cardiac growth and function..NatMed.2000,6:1221-1227.
16 Karliner JS, Kariya T, and Simpson PC. Effects of pertussis toxin on al-agonist-mediated phosphatidylinositide turnover and myocardial cell hypertrophy in neonatal rat myocytes. Experientia.1990,46:81-84.
17 Fu M, Xu S, Zhang J, Pang Y, Liu N, Su J, Tang C. Involvement of calcineurin in angiotensin Ⅱ-induced cardiomyocyte hypertrophy and cardiac fibroblast hyperplasia of rats. Heart and Vessels.1999,14:283-188.
18 Zhu W, Zou Y, Shiojima L, Kudoh S, Aikawa R, Hayashi D, Mizukami M, Toko H, Shibasaki F, Yazaki Y, Nagai R, Komuro I. Ca2+/calmodulin-dependent kinase Ⅱ and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy. J Biol Chem.2000,275:15239-15245.
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21 Carnicelli V, Frascarelli S, Ghelardoni S, Ronca-Testoni S, Zucchi R. Short-term effects of pressure overload on the expression of genes involved in calcium homeostasis. Mol Cell Biochem.2008,313:29-36.
22 van der Velden J, Merkus D, Klarenbeek BR, James AT, Boontje NM, Dekkers DH, Stienen GJ, Lamers JM, Duncker DJ. Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardiol infarction. Circ Res.2004,95:e85-e95.
23 Highighi K, Schmidt AG, Hiot BD, Brittsan AG, Yatani A, Lester JW, Zhai J, Kimura Y, Dorn GW 2nd, MacLennan DH, Kranias EG. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractle failure. J Biol Chem.2001,276:24145-24152.
24 Aramburu J, Rao A, Klee CB. Calcineurin:from structure to function. Curr Top Cell Regul.2000,36:237-295.
25 Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science.1997,276:1404-1407.
26 McKinsey TA, Zhang CL, Olson EN. MEF2:a calcium-dependent regulator of cell dividion, differentiation and death. Trends Biochem Sci.2002,27:40-47.
27 Molkentin JD, Markham BE:Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo. J Biol Chem.1993,268:19512-19520.
28 Bhavsar PK, Dellow KA, Yacoub MH, Brand NJ, Barton PJ. Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. J Mol Cell Cardiol.2000,32:95-108.
29 Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science.1997,276:1404-1407.
30 Zhao Z, Geng J, Ge Z, Wang W, Zhang Y, Kang W. Activation of ERK5 in angiotensin II-induced hypertrophy of human aortic smooth muscle cells. Mol Cell Biochem.2009,322:171-8.
31 Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, Overbeek P,
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1 Frey N, McKinsey TA, Olson EN. Decoding calcium signals involved in cardiac growth and function. NatMed,2000,6:1221-1227.
2苏丹,许兵,石海莲,吴大正,戴亚蕾.黄芪注射液对左室肥厚大鼠心肌钙超载及肌浆网钙泵表达的影响.中国中药杂志.2008,33：1724-1727
3 Karliner JS, Kariya T, and Simpson PC. Effects of pertussis toxin on al-agonist-mediated phosphatidylinositide turnover and myocardial cell hypertrophy in neonatal rat myocytes. Experientia.1990.46:81-84.
4 Fu M, Xu S, Zhang J, Pang Y, Liu N, Su J, Tang C. Involvement of calcineurin in angiotensin II-induced cardiomyocyte hypertrophy and cardiac fibroblast hyperplasia of rats. Heart and Vessels.1999,14:283-288.
5 Zhu W, Zou Y, Shiojima L, Kudoh S, Aikawa R, Hayashi D, Mizukami M, Toko H, Shibasaki F, Yazaki Y, Nagai R, Komuro I. Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy. J Biol Chem.2000,275:15239-15245.
6 Chu G, Lester JW, Young KB, Luo W, Zhai J, Kranias EG. A single site (Ser16) phosphorylation in phospholamban is sufficient in mediating its maximal cardiac responses to beta-agonists. J Biol Chem.2000,275:38938-38943
7 Koyanagi T, Wong LY, Inagaki K, Petrauskene OV, Mochly-Rosen D. Alteration of gene expression during progression of hypertension-induced cardiac dysfunction in rats. Am J Physiol Heart Circ Physiol.2008,295:H220-H226.
8 Carnicelli V, Frascarelli S, Ghelardoni S, Ronca-Testoni S, Zucchi R. Short-term effects of pressure overload on the expression of genes involved in calcium homeostasis. Mol Cell Biochem.2008,313:29-36.
9 van der Velden J, Merkus D, Klarenbeek BR, James AT, Boontje NM, Dekkers DH, Stienen GJ, Lamers JM, Duncker DJ. Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardiol infarction. Circ Res.2004,95:e85-e95.
10 Highighi K, Schmidt AG, Hiot BD, Brittsan AG, Yatani A, Lester JW, Zhai J,
Kimura Y, Dorn GW 2nd, MacLennan DH, Kranias EG. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractle failure. J Biol Chem.2001,276:24145-24152.
11 Arnaudeau S, Frieden M, Nakamura K, Castelbou C, Michalak M, Demaurex N. Calreticulin differentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria. J Biol Chem.2002,277:46696-46705.
12 John LM, Lechleiter JD, Camacho P. Differential modulation of SERCA2 isoforms by calreticulin. J Cell Biol.1998,142:963-973.
13 Aramburu J, Rao A, Klee CB. Calcineurin:from structure to function. Curr Top Cell Regul.2000,36:237-295.
14 Kell CB, Ren H, Wang X. Regulation of tile calmodulin-stimulated protein phosphatase, calcineurin. J Biol Chem.1998,273:13367.
15 Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science.1997,276:1404-1407.
16 McKinsey TA, Zhang CL, Olson EN. MEF2:a calcium-dependent regulator of cell dividion, differentiation and death. Trends Biochem Sci.2002,27:40-47.
17 Molkentin JD, Markham BE:Myocyte-specific enhancer-binding factor (MEF-2) regulates alpha-cardiac myosin heavy chain gene expression in vitro and in vivo. J Biol Chem.1993,268:19512-19520.
18 Bhavsar PK, Dellow KA, Yacoub MH, Brand NJ, Barton PJ. Identification of cis-acting DNA elements required for expression of the human cardiac troponin I gene promoter. J Mol Cell Cardiol.2000,32:95-108.
19 Lin Q, Schwarz J, Bucana C, Olson EN. Control of mouse cardiac morphogenesis and myogenesis by transcription factor MEF2C. Science.1997,276:1404-1407.
20 Zhao Z, Geng J, Ge Z, Wang W, Zhang Y, Kang W. Activation of ERK5 in angiotensin Ⅱ-induced hypertrophy of human aortic smooth muscle cells. Mol Cell Biochem.2009,322:171-178.
21 Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, Overbeek P, Richardson JA,Grant SR, Olson EN. CaM kinase signaling induces cardiac hypertrophy and actives the MEF2 transcription factor in vivo. J Clin Invest.2000, 105:1395-1406.
22 Prasad AM, Inesi G Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes. Am J Physiol Cell Physiol.2009,296:C992-C1002.
23 Sakuma K, Akiho M, Nakashima H, Nakao R, Hirata M, Inashima S, Yamaguchi A, Yasuhara M. Cyclosporin A modulates cellular localization of MEF2C protein and blocks fiber hypertrophy in the overloaded soleus muscle of mice. Acta Neuropathol.2008,115:663-674.