ALDH2在异氟烷预处理抗MIRI中的作用及其调控机制研究
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摘要
研究目的：通过构建抑制ALDH2基因表达的腺病毒和ALDH2持续活化的突变体腺病毒，来证实ALDH2在IsoP诱导的心脏保护中所起到的关键作用，并确定ALDH2表达调控在功能上的重要性,
研究方法(1)根据ALDH2基因序列及siRNA的设计原则，参考Cortinez,G的文献，设计小分子干扰RNA的序列，以ALDH2为靶基因设计并合成小发卡结构两端配对的siRNA寡核苷酸链，再经变性，复性后形成双链ALDH2-siRNA。采用DNA重组技术，将ALDH2-siRNA与线性化RetroQ载体连接，酶切鉴定后进行扩增，然后将Rat-ALDH2-siRNA从RetroQ-Rat-ALDH2(siRNA)上切下连接到pShuttle-Basic-ERFP重组穿梭载体，得到pShuttle-RFP-Rat-ALDH2-siRNA重组穿梭质粒，将pShuttle-RFP-Rat-ALDH2-siRNA转移到pAdeno载体上，得到pAdeno-RFP-Rat-ALDH2-siRNA病毒质粒。(2)以前的研究表明：持续活化的ALDH2487位氨基酸必须是谷氨酸而不是赖氨酸，Thr185、Thr412和Ser279需磷酸化。因此，我们通过在野生型大鼠ALDH2的cDNA引入突变Lys487Glu，Thr185Asp，Thr412Asp和Ser279Asp，获得了持续活化突变型ALDH2（CA-ALDH2），用DNA重组技术将CA-ALDH2基因连接到pShuttle-CMV-RFP重组穿梭载体，得到pShuttle-CA-ALDH2-RFP重组穿梭质粒；将pShuttle-CA-ALDH2-RFP转移到pAdeno载体上，得到pAdeno-CA-ALDH2病毒质粒。
研究结果：(1)成功构建了以ALDH2基因为靶点的pAdeno-RFP-Rat-ALDH2-siRNA病毒载体，经酶切鉴定，所构载体无基因突变，结果与预期符合。(2)通过定点突变技术成功构建了pAdeno-CA-ALDH2病毒载体，经酶切鉴定，所构载体无基因突变，为下一步实验奠定了基础。
研究结论：成功构建了pAdeno-RFP-Rat-ALDH2-siRNA病毒载体,Western blot结果表明该siRNA能很好的抑制ALDH2基因在大鼠心肌细胞中的表达；成功构建了pAdeno-CA-ALDH2病毒载体，Western blot结果表明该基因能在心肌细胞中持续表达。
研究目的：以构建好的ALDH2干扰型和永久激活型腺病毒载体感染新生大鼠的心肌细胞，在基因水平特异性激活或抑制ALDH2，研究ALDH2在IsoP抗心肌细胞凋亡中的作用，并探索其抗凋亡机制。
研究方法：将原代培养的1日龄新生SD大鼠心肌细胞随机同样地分成如下不同的实验组：空质粒感染同时异氟烷处理和未处理组；Adeasy-Si-ALDH2感染同时异氟烷处理和未处理组以及Adeasy-CA-ALDH2感染同时异氟烷处理和未处理组。使用含0.5mM异氟烷（大约1.0肺泡最小浓度）的0.5％FBS的DMEM培养细胞5分钟，进行IsoP。在缺氧处理前迅速除去含异氟烷的培养基并用PBS洗涤细胞。低氧室中培养离体的新生大鼠心肌细胞24h后供氧12h以模拟I/R制备H/R模型。H/R结束后采用免疫印迹法(WesternBlot)检测各实验组凋亡通路蛋白Cleaved caspase-3和FL-caspase-3的表达、用Caspase-3比色测定试剂盒检测细胞裂解产物中的caspase-3酶活性、用TUNEL法检测心肌细胞凋亡水平以及用Cytotoxicity Detection Kit(LDH)检测心肌细胞培养基中的LDH释放量。综合上述指标评估ALDH2下调或活化在IsoP抗心肌细胞凋亡损伤中的作用。
研究结果：IsoP通过激活AlDH2缓解了体外H/R损伤诱导的心肌细胞凋亡：缺氧24h复氧12h后，与未经H/R处理的心肌细胞相比，H/R处理显著升高了心肌细胞的凋亡指数（图1B）、凋亡通路蛋白酶Cleaved Caspase3的相对表达（图1C）、Caspase3活性（图1D）和LDH释放（图1E），TUNEL染色，可见DNA碎片增加，即明显的心肌细胞凋亡。IsoP显著降低了Ad-RFP感染组H/R诱导的心肌细胞的凋亡指数、Cleaved Caspase-3的相对表达、Caspase3活性和LDH浓度的升高（P﹤0.05，图1B-E）。然而在Ad-Si-ALDH2感染组中，由Ad-Si-ALDH2介导ALDH2基因下调使ALDH2蛋白表达水平降低的情况下（图1A），IsoP不能降低H/R诱导的心肌细胞的凋亡指数、Cleaved Caspase-3的相对表达、Caspase-3活性和LDH释放的显著升高（P﹥0.05，图1B-E），即Ad-Si-ALDH2介导ALDH2基因下调后取消了IsoP的上述心脏保护作用，IsoP与未处理组无显著差异。上述结果表明：IsoP显著抑制了H/R诱导的心肌细胞凋亡，而ALDH2基因下调则抑制了IsoP诱导的抗心肌细胞凋亡的作用。这一结果支持我们的假设即ALDH2可能在IsoP诱导的心肌保护中发挥关键作用。免疫印迹分析表明，与Ad-RFP感染的细胞组相比，Ad-CA-ALDH2-RFP感染的细胞组中的ALDH2水平大幅度增加（图2A）。持续活化的ALDH2显著抑制H/R引起的TUNEL阳性染色的增加，Caspase-3活性的增加和LDH的释放（P﹤0.05，图2B-E，）。而在Ad-CA-ALDH2介导ALDH2持续活化的情况下，再用IsoP则不能进一步增强上述心脏保护作用,即IsoP与未处理组无差异。这说明了活化的ALDH2（即磷酸化的ALDH2）参与IsoP诱导的心脏保护作用。
研究结论：IsoP通过激活AlDH2缓解了体外H/R损伤诱导的心肌细胞凋亡。ALDH2基因下调,H/R后心肌细胞凋亡数目大大增加，持续活化的ALDH2（即ALDH2基因上调）显著抑制H/R引起的心肌细胞凋亡，这表明活化的ALDH2参与了IsoP诱导的心肌保护作用。这一结果支持我们的假设即磷酸化ALDH2可能在IsoP诱导的心肌保护中发挥关键作用。ALDH2的抗心肌细胞凋亡作用的机制可能与其抑制Caspase3的激活从而抑制凋亡通路的激活有关。
研究目的：通过ALDH2激动剂及抑制剂，在药理水平激动或者抑制ALDH2，研究内源性ALDH2及其特异位点磷酸化在IsoP抵抗在体MIRI中的作用。
研究方法：采用结扎冠状动脉左前降支(LAD)40分钟之后进行120分钟再灌注处理制备雄性SD大鼠心肌I/R模型。为了证实异氟烷诱导的ＡＰＣ作用，从大鼠稳定期开始，以最小肺泡浓度1.0（2.1%）的异氟烷开始持续吸入30分钟，之后进行30分钟洗脱，再进行冠状动脉闭塞处理。根据不同的处理方法将大鼠随机分配到下列各组：假手术对照组和IsoP的假手术对照组：无/有IsoP，开胸分离冠状动脉左前降支，仅在LAD下穿线，但不阻断血流,持续160分钟(每组5只)；缺血再灌注组和IsoP的缺血再灌注组：无/有IsoP，可逆性的结扎LAD造成区域性心肌缺血40分钟，再灌注120分钟(每组5只)；为了评估ALDH2磷酸化在异氟烷诱导的心脏保护中的作用，在IsoP和无IsoP缺血处理前5分钟分别给予ALDH2的激活剂Alda-44（40μM）（每组8只），和ALDH2抑制剂cyanamide（5mM），（每组8只）。再灌注结束后，取出心脏采用伊文思蓝和TTC双重染色法测定心肌梗死面积。取出心脏前采集5ml血样，LDH和CK-MB的浓度用全自动生化分析仪7600及商用试剂盒进行测定；采用免疫印迹法测定信号通路蛋白(磷酸化的ALDH2and总ALDH2)的表达。
研究结果：(1)IsoP诱导的心脏保护作用：与假手术对照组相比，结扎LAD引起区域性心肌缺血40分钟然后进行120分钟再灌注使I/R组大鼠的血清LDH和CK-MB浓度和心梗面积显著增加(P<0.01)。而IsoP诱导的APC作用显著地降低了I/R所导致的心肌细胞LDH和CK-MB释放且大幅度地减少了I/R-诱导的心肌梗死面积（P<0.05）见图1；(2)ALDH2的磷酸化参与IsoP诱导的心脏保护:与I/R对照组相比，IsoP后使心肌细胞中磷酸化的ALDH2浓度显著升高（P<0.01），即缺血前IsoP增加了ALDH2的磷酸化。而ALDH2酶抑制剂cyanamide显著抑制了异氟烷诱导的ALDH2磷酸化后的激活效应（P<0.05）。使用ALDH2的直接激活剂Alda-44大幅度地增加了心肌细胞中ALDH2的磷酸化水平（P<0.01），但Alda-44并没有使IsoP诱导的ALDH2磷酸化效应进一步增强（图2A）。与ALDH2磷酸化相一致，我们观察到IsoP和使用Alda-44均显著减少了I/R引起的心肌细胞LDH和CK-MB向血浆的泄漏并减少了心梗面积（P<0.01，图2B-D）。IsoP和同时使用Alda-44不能进一步增强这种降低，即二者无叠加效应。然而，IsoP诱导的减少LDH和CK-MB释放和减少了心梗面积效应可明显被ALDH2抑制剂氰胺所阻断（P<0.01，图2B-D）。这些研究结果表明IsoP介导的心脏保护主要是由激活ALDH2所引起。
研究结论：IsoP诱导的APC作用显著减少了大鼠心脏I/R损伤所致的LDH和CK-MB释放，通过增加ALDH2的磷酸化减少了I/R-诱导的心肌梗死面积。我们推测IsoP介导的心脏保护可能主要是由激活ALDH2所引起。
研究目的：通过WesternBlot检测PKC两种亚型（PKCε和PKCδ）的线粒体转位，确定PKC两种亚型在IsoP抗MIRI中的活化情况；通过加入PKC两种亚型的抑制剂，研究PKC双亚型在ALDH2介导的IsoP抗MIRI中的作用。从而探索PKC信号通路与ALDH2分子的内源性信号转导途径与分子调控机制。
研究方法：(1)为了验证PKCε参与ALDH2的磷酸化，在IsoP和无IsoP缺血处理前5分钟给予雄性SD大鼠PKCε抑制剂PKCεV1-2（1μM）（每组8只），分别测定各实验组（I/R组、I/R+IsoP组、I/R+εV1-2组和I/R+IsoP+εV1-2组）大鼠的血清LDH和CK-MB的浓度，并用伊文思蓝-TTC双染色法测定心梗面积，并计算出心梗面积的百分比。Westernblot测定信号通路蛋白（Phos-ALDH2和Total-ALDH2、Mito-PKCε与Total-PKCε）的表达；(2)为了探讨IsoP对PKCδ的活化及向线粒体转位的影响，分别测定各实验组（SHAM组、I/R组、SHAM+IsoP组和I/R+IsoP组）的Mito-PKCδ和Total-PKCδ的表达量。（3）为了探讨PKCδ在ALDH2磷酸化中的作用，在IsoP和无IsoP缺血处理前5分钟给予PKCδ的抑制剂Rottlerin（1μM）（每组8只）。分别测定各实验组（I/R组、I/R+IsoP组、I/R+rottlerin组和I/R+IsoP+rottlerin组）大鼠血清中LDH和CK-MB的浓度。Westernblot测定信号通路蛋白（Phos-ALDH2和Total-ALDH2）的表达。研究结果：(1)PKCε参与了IsoP诱导的ALDH2的磷酸化和心脏保护：PKCε转位到线粒体后使ALDH2磷酸化是保护心脏免受I/R损伤的必需步骤。与未经
IsoP的I/R对照组相比，IsoP后心肌细胞线粒体中的PKCε浓度显著升高（P<0.01）同时伴有心肌细胞中磷酸化的ALDH2浓度显著升高（P<0.01），而PKCε抑制剂εV1-2能显著抑制这种升高（P<0.01）。在此，我们证明：IsoP导致PKCε在线粒体中浓度升高且伴随着ALDH2的磷酸化。IsoP介导的ALDH2的磷酸化被PKCε抑制剂εV1-2所抑制。由于PKCε在线粒体中的转位迅速发生，导致细胞质基质PKCε水平相应下降，因为细胞总的PKCε水平没有发生变化（图1A），因此我们的数据表明IsoP使PKCε动态转位到线粒体上以应对I/R引起的损伤。与PKCε转运到线粒体的情形相对应，PKCε抑制剂εV1-2取消了IsoP诱导的减少LDH和CK-MB释放效应以及减少心肌梗死面积的效应(图1B-D)；(2)IsoP减少了心肌细胞的PKCδ的活化及向线粒体转位：我们的数据表明：与假手术对照组相比，I/R导致心肌细胞线粒体中的PKCδ浓度显著增高（P﹤0.01），IsoP后大大减少了心肌细胞中的PKCδ向线粒体转位使心肌细胞线粒体中的PKCδ浓度显著降低（P﹤0.01）（图2A-B）（3）PKCδ向线粒体转位减少参与了IsoP诱导的心脏保护：IsoP减少了PKCδ从胞浆转位到线粒体中，PKCδ抑制剂rottlerin显著抑制了I/R导致的心肌细胞ALDH2磷酸化水平的降低（图3A-B）以及LDH和CK-MB释放（图3C-D），其效果与IsoP相当免疫印迹分析结果表明：使用PKCδ抑制剂rottlerin抑制了PKCδ的活性后,无论是否施用IsoP，I/R损伤后ALDH2磷酸化水平都显著升高。类似地，IsoP使PKCδ的活性被抑制从而使PKCδ线粒体移位减少以应对I/R引起的心肌损伤。研究结论：IsoP导致PKCε在线粒体中的浓度显著升高且伴随着ALDH2的磷
酸化，同时减少PKCδ的线粒体移位，通过双向调节PKC双亚型（PKCε和PKCδ）通路在心肌I/R过程中的活化水平，从而进一步激活了ALDH2，抵抗了心肌I/R损伤。即IsoP诱导的抗心肌I/R损伤机制可能是通过激活PKC（PKCε和PKCδ）-ALDH2信号通路实现的。
Objective:Confirm the critical role of ALDH2in isoflurane-induced cardioprotection and detect the functional significance of manipulating ALDH2expression by construction of ALDH2knockdown adenovirus and constitutively active ALDH2mutant adenovirus.
Methods (1) according to the sequence of ALDH2and the principle of SiRNA,we designed a siRNA refer to Cortinez, G's paper, target the ALDH2gene we synthesis small hairpin siRNA, after denature and refolding,formed double strand ALDH2-siRNA.Using the DNA recombination technology,we linked ALDH2-siRNA and lined RetroQ vector. After Identification by restriction endonuclease digestion and replication, we cut the Rat-ALDH2-SiRNA from RetroQ-Rat-ALDH2and link it to pShuttle-Basic-ERFP shuttle vector, get the pShuttle-RFP-Rat-ALDH2-siRNA vector. Finally we transfer pShuttle-RFP-Rat-ALDH2-siRNA to pAdeno vector to get pAdeno-RFP-Rat-ALDH2-siRNA vector.(2) Previous studies demonstrated that the constitutively active ALDH2amino acid487must be Glu not Lys, and Thr185, Thr412and Ser279must be constitutively phosphorylated. Accordingly, we obtained constitutively active mutant ALDH2(CA-ALDH2) by nucleotide substitutions leading to the mutations Lys487Glu, Thr185Asp, Thr412Asp, and Ser279Asp introduced into the wild-type rat ALDH2cDNA.Using the DNA recombination technology, We linked CA-ALDH to pShuttle-CMV-RFP shuttle vector,the pShuttlet-CA-ALDH2-RFP vector. We transfer pShuttle-CA-ALDH2-RFP to pAdeno vector to get pAdeno-CA-ALDH2-RFP vector.
Results:(1)Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector, after Identification by restriction endonuclease digestion, there is no mutation in the sequence, which is in our expectation.(2) Using the site-mutation technology we constructed pAdeno-CA-ALDH2vector after Identification by restriction endonuclease digestion, and got the right sequence, which Laid the foundation for our next experiments.
Conclusions:Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector,Western blot analysis showed siRNA can inhibite ALDH2gene expression in rat Myocardial cells;Successfully constructed pAdeno-CA-ALDH2vector,Western blot analysis showed the CA-ALDH2can expressed in Myocardial cells quite well.
Objective:Elucidate the role of ALDH2and in anti-apoptosis effect induced by isoflurane pretreatment by implementing specific activation or suppression on ALDH2gene, where neonatal rat cardiomyocytes were infected by pre-prepared ALDH2knockdown and constitutively active mutant adenovirus vectors.
Methods:primary cultured one-day old neonatal SD rat cardiomyocytes were randomly and homogeneously divided into different experimental groups as follows: vector-infected with and without isoflurane, Adeasy-Si-ALDH2-treated with and without isoflurane; vector-infected with and without isoflurane, Adeasy-CA-ALDH2-treated with and without isoflurane. Exposure to isoflurane was performed by incubating the cells for5minutes in0.5mM isoflurane (approximately1.0minimum alveolar concentration) in0.5%FBS DMEM. The isoflurane-containing medium was removed immediately before the onset of hypoxic conditions and the cells were washed with phosphate-buffered saline(PBS). Simulated I/R was achieved by culturing the Isolated neonatal cardiomyocytes in0.5%FBS DMEM in a hypoxia chamber, saturated with5%CO2/95%N2at37℃for24h and following reoxygenation for12h using0.5%FBS DMEM in the normal incubating condition. At the end of H/R, the apoptosis pathway proteins(Cleaved caspase-3and full-length caspase-3) in different cell groups were determined by Western Blot analysis; the caspase-3enzyme activity of cell lysate in different groups was detected by Caspase-3Colorimetric assay kit; apoptosis index was performed using terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) with an in situ cell death detection kit, according to the manufacturer's protocol for cultured cells and the lactate dehydrogenase(LDH)activity released from the cytosol of damaged cells into the culture medium was measured by Cytotoxicity Detection Kit (LDH, Roche). The role of ALDH2downregulation or activation in isoflurane-induced myocardial anti-apoptotic effect was evaluated by the values above.
Results:Isoflurane preconditioning attenuated cardiomyocytes apoptosis induced by H/R injury in vitro:After24h of hypoxia followed by12h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry and caspase3activity in the vector control(Ad-RPF) group (Figure1B-E). Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure1B-E). However, when ALDH2was downregulated (Figure1A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level, more intense cleaved caspase-3staining, caspase3activity and LDH release (P>0.05, FigurelB-E) were observed, which supports the hypothesis that ALDH2might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure2A). Constitutively active ALDH2induced by Ad-CA-ALDH2significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure2B-E), which were not further increased by isoflurane treatment, i.e. no significant difference existed between control and isoflurane-pretreatment group. This suggests that activated (or phosphorylated) ALDH2involves isoflurane-induced cardioprotection.
Conclusions:Isoflurane preconditioning alleviated in vitro H/R injury by activation of ALDH2. Downregulated ALDH2gene leads to a great increase in apoptotic cardiomyocytes. Constitutively activated ALDH2(upregulated ALDH2gene) results in a significant inhibition of H/R induced cardiomyocytes apoptosis, which indicates that activated ALDH2involves isoflurane-induced cardioprotection. This result supports our hypothesis that phosphorylated ALDH2may play a critical role in isoflurane-induced cardioprotection. The anti-apoptotic mechanism of ALDH2may involves inhibition of caspase-3and hence inhibition of apoptotic pathway.
Objective:Reveal the role and mechanism of endogenic ALDH2and its phosphorylation at specific site in anti-apoptosis effect induced by isoflurane pretreatment by means of pharmacologic activation or inhibition of ALDH2in vivo.
Methods:The acute myocardial I/R injury model was performed by LAD ligation. To confirm isoflurane-induced APC, a minimal alveolar concentration of isoflurane of1.0(2.1%) was started at the end of the stabilization period and administered for30minutes, followed by30minutes of washout before coronary occlusion. Rats were randomly assigned to one of the following groups subjected to different protocols:Sham groups, non-ischemic control group of sham-operated rats without and with isoflurane:rats were opened chest without ligating LAD for160minutes (n=5,respectively); I/R group andIsoP+I/R group:40minutes of myocardial ischemia and120minutes of reperfusion without and with isoflurane (n=5,respectively). To evaluate the role of ALDH2in isoflurane-induced cardioprotection, the direct activator of ALDH2, Alda-44(40μM), was given5minutes prior to ischemia in the groups without and with isoflurane (n=8, respectively), and the ALDH2inhibitor, cyanamide (5mM), was given without and with isoflurane (5minutes prior to isoflurane)(n=8, respectively). At the end of the reperfusion, the heart was removed and infarct size(IS) and area at risk(AAR) were assayed by Evans Blue and TTC staining. Before removing the heart,5ml blood samples were taken. Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assayed using commercial kits by an automatic analyzer7600; signal pathway proteins (Phos-ALDH2and Total-ALDH2in different groups) were assayed by Western Blot analysis.
Results:(1) Cardioprotective effect induced by Isoflurane preconditioning: Regional myocardial ischemia for40minutes by LAD ligation followed by120minutes of reperfusion markedly increased the leakage of LDH, CK-MB and myocardial infarctsize compared to sham controls(P<0.01). Isoflurane-induced APC significantly reduced the I/R-induced increase in LDH, CK-MB release in rat heart and substantially decreased I/R-induced myocardial infarct size (P<0.05). As shown in Figure1.(2) Phosphorylation of ALDH2participated in cardioprotection induced by IsoP:Pretreatment with isoflurane prior to ischemia increased the phosphorylation of ALDH2compared to I/R groups (P<0.01). The ALDH2inhibitor, cyanamide, significantly inhibited isoflurane-induced activation of ALDH2(P<0.05). The direct activator of ALDH2, Alda-44, substantially increased the phosphorylation of ALDH2P<0.01), but did not enhance the phosphorylation of ALDH2by isoflurane (图2A) Consistent with ALDH2phosphorylation,we observed that isoflurane and Alda-44markedly attenuated I/R-induced leakage of LDH and CK-MB in plasma, as well as myocardial infarct size (P<0.01,图2B-D) However, the isoflurane-induced decrease in LDH and CK-MB release, and reduction of infarct size was significantly blocked by the ALDH2inhibitor cyanamide (P<0.01, Figure2B-D). These findings suggest that isoflurane-mediated cardioprotection is mainly mediated by activation of ALDH2.
Conclusions:LDH and CK-MB leakage from rat heart I/R injury is significantly reduced by APC effect by isoflurane preconditioning, with a decrease in infarct size caused by upregulation of ALDH2phosphorylation. We suggest isoflurane-induced cardioprotective effect may essentially result from ALDH2activation.
Objective:Measure the activity of two PKC subtypes (PKCε and PKCδ) in isoflurane pretreatment against myocardial I/R injury by detection of mitochondrial translocation of both subtypes with Western blotting. Identify the role of both subtypes in ALDH2-mediated isoflurane pretreatment by application of inhibitors of both subtypes. Explore endogenic PKC and ALDH2signaling pathway and its molecular regulation mechanism.
Methods:(1) To verify that PKCε participates in the phosphorylation of ALDH2, the PKCε inhibitor, PKCε v1-2(1μM), was given5minites prior to ischemia without and with isoflurane (n=8, respectively,).Analyze the concentration of LDH and CK-MB in the rat Serum. Evans Blue-TTC stainning to analyze infarct area in different groups (I/R, I/R+IsoP, I/R+εvl-2and I/R+IsoP+εvl-2group). Signaling pathway proteins(Phos-ALDH2, Total-ALDH2, Mito-PKCε and Total-PKCε in Four different groups) were assayed by Western Blot analysis.(2) To identify the effect of isoflurane preconditioning on PKCδ activation and translocation, we measured mito-PKCδ and total PKCδ expression in all groups (Sham, I/R, Sham+IsoP and I/R+IsoP group).(3) To identify the role PKCδ plays in phosphorylation of ALDH2, the PKCδ inhibitor, Rottlerin(1μM), was given5minites prior to ischemia without and with isoflurane (n=8).Analyze the concentration of LDH and CK-MB in the Serum. Signaling pathway proteins(Phos-ALDH2, Total-ALDH2in Four different groups) were assayed by Western Blot analysis.
Results:(1) PKCε is involved in isoflurane-induced phosphorylation of ALDH2and cardioprotection:PKCε translocation to mitochondria and then phosphorylation of ALDH2is required to protect the heart from I/R injury. Compared with control group without isoflurane preconditioning, isoflurane pretreatment significantly enhanced mitochondrial PKCε concentration in cardiomyocytes (P<0.01), accompanied with significant increase in phosphorylated ALDH2in cardiomyocytes (P<0.01), whileεV1-2, a PKCε inhibitor, could significantly inhibit this effect (P<0.01). Here we demonstrate that pretreatment with isoflurane resulted in elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2. Isoflurane-induced phosphorylation of ALDH2was inhibited by the PKCε inhibitor, PKCεV1-2. Because mitochondrial translocation of PKCε occurs rapidly, with a corresponding decline in cytosolic PKCε levels, and because the total cellular PKCε levels do not change (FigurelA), our data suggest that pretreatment with isoflurane enables dynamic mitochondrial translocation of PKCε in response to I/R. Consistent with PKCε translocation to mitochondria, PKCεV1-2had a detrimental effect on isoflurane-induced attenuation of LDH and CK-MB leakage and the decrease in myocardial infarct size (Figure1B-D);(2) Pretreatment with isoflurane resulted in decreased PKCδ activity and mitochondrial levels in cardiomyocytes. Our data shows that compared to sham groups, I/R led to a significant increase in PKCδ levels in mitochondria of cardiomyocytes (P<0.01), Isoflurane preconditioning greatly inhibits mitochondria translocation of PKCδ in cardiomyocytes, and significantly decreases mitochondria PKCδ concentration in cardiomyocytes (P<0.01)(Figure2A-B).(3) Decreased level of PKCδ translocation to mitochondria involves isoflurane preconditioning-induced cardioprotection:Isoflurane-afforded inhibition of ALDH2phosphorylation level decrease (Figure3A-B) and LDH and CK-MB release (Figure3C-D) by I/R was mimicked by Rottlerin. Western blot showed that inhibition of PKCδ significantly elevated phosphorylation of ALDH2regardless of isoflurane preconditioning. Similarly, isoflurane preconditioning inhibits PKCδ activity, thus inhibiting its mitochondria translocation to act against I/R-induced myocardial injury.
Conclusions:Pretreatment with isoflurane resulted in significantly elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2, in the same time attenuated translocation of PKCδ. The bi-directional regulation of PKC two subtypes activation level during I/R would further activate ALDH2, and guarantee resistance to ischemia-reperfusion injury. Isoflurane preconditioning may activate PKC (PKCεand PKCδ)-ALDH2signaling pathway to induce protective effect against myocardial I/R injury.
研究方法(1)根据ALDH2基因序列及siRNA的设计原则，参考Cortinez,G的文献，设计小分子干扰RNA的序列，以ALDH2为靶基因设计并合成小发卡结构两端配对的siRNA寡核苷酸链，再经变性，复性后形成双链ALDH2-siRNA。采用DNA重组技术，将ALDH2-siRNA与线性化RetroQ载体连接，酶切鉴定后进行扩增，然后将Rat-ALDH2-siRNA从RetroQ-Rat-ALDH2(siRNA)上切下连接到pShuttle-Basic-ERFP重组穿梭载体，得到pShuttle-RFP-Rat-ALDH2-siRNA重组穿梭质粒，将pShuttle-RFP-Rat-ALDH2-siRNA转移到pAdeno载体上，得到pAdeno-RFP-Rat-ALDH2-siRNA病毒质粒。(2)以前的研究表明：持续活化的ALDH2487位氨基酸必须是谷氨酸而不是赖氨酸，Thr185、Thr412和Ser279需磷酸化。因此，我们通过在野生型大鼠ALDH2的cDNA引入突变Lys487Glu，Thr185Asp，Thr412Asp和Ser279Asp，获得了持续活化突变型ALDH2（CA-ALDH2），用DNA重组技术将CA-ALDH2基因连接到pShuttle-CMV-RFP重组穿梭载体，得到pShuttle-CA-ALDH2-RFP重组穿梭质粒；将pShuttle-CA-ALDH2-RFP转移到pAdeno载体上，得到pAdeno-CA-ALDH2病毒质粒。
研究结果：(1)成功构建了以ALDH2基因为靶点的pAdeno-RFP-Rat-ALDH2-siRNA病毒载体，经酶切鉴定，所构载体无基因突变，结果与预期符合。(2)通过定点突变技术成功构建了pAdeno-CA-ALDH2病毒载体，经酶切鉴定，所构载体无基因突变，为下一步实验奠定了基础。
研究结论：成功构建了pAdeno-RFP-Rat-ALDH2-siRNA病毒载体,Western blot结果表明该siRNA能很好的抑制ALDH2基因在大鼠心肌细胞中的表达；成功构建了pAdeno-CA-ALDH2病毒载体，Western blot结果表明该基因能在心肌细胞中持续表达。
研究目的：以构建好的ALDH2干扰型和永久激活型腺病毒载体感染新生大鼠的心肌细胞，在基因水平特异性激活或抑制ALDH2，研究ALDH2在IsoP抗心肌细胞凋亡中的作用，并探索其抗凋亡机制。
研究方法：将原代培养的1日龄新生SD大鼠心肌细胞随机同样地分成如下不同的实验组：空质粒感染同时异氟烷处理和未处理组；Adeasy-Si-ALDH2感染同时异氟烷处理和未处理组以及Adeasy-CA-ALDH2感染同时异氟烷处理和未处理组。使用含0.5mM异氟烷（大约1.0肺泡最小浓度）的0.5％FBS的DMEM培养细胞5分钟，进行IsoP。在缺氧处理前迅速除去含异氟烷的培养基并用PBS洗涤细胞。低氧室中培养离体的新生大鼠心肌细胞24h后供氧12h以模拟I/R制备H/R模型。H/R结束后采用免疫印迹法(WesternBlot)检测各实验组凋亡通路蛋白Cleaved caspase-3和FL-caspase-3的表达、用Caspase-3比色测定试剂盒检测细胞裂解产物中的caspase-3酶活性、用TUNEL法检测心肌细胞凋亡水平以及用Cytotoxicity Detection Kit(LDH)检测心肌细胞培养基中的LDH释放量。综合上述指标评估ALDH2下调或活化在IsoP抗心肌细胞凋亡损伤中的作用。
研究结果：IsoP通过激活AlDH2缓解了体外H/R损伤诱导的心肌细胞凋亡：缺氧24h复氧12h后，与未经H/R处理的心肌细胞相比，H/R处理显著升高了心肌细胞的凋亡指数（图1B）、凋亡通路蛋白酶Cleaved Caspase3的相对表达（图1C）、Caspase3活性（图1D）和LDH释放（图1E），TUNEL染色，可见DNA碎片增加，即明显的心肌细胞凋亡。IsoP显著降低了Ad-RFP感染组H/R诱导的心肌细胞的凋亡指数、Cleaved Caspase-3的相对表达、Caspase3活性和LDH浓度的升高（P﹤0.05，图1B-E）。然而在Ad-Si-ALDH2感染组中，由Ad-Si-ALDH2介导ALDH2基因下调使ALDH2蛋白表达水平降低的情况下（图1A），IsoP不能降低H/R诱导的心肌细胞的凋亡指数、Cleaved Caspase-3的相对表达、Caspase-3活性和LDH释放的显著升高（P﹥0.05，图1B-E），即Ad-Si-ALDH2介导ALDH2基因下调后取消了IsoP的上述心脏保护作用，IsoP与未处理组无显著差异。上述结果表明：IsoP显著抑制了H/R诱导的心肌细胞凋亡，而ALDH2基因下调则抑制了IsoP诱导的抗心肌细胞凋亡的作用。这一结果支持我们的假设即ALDH2可能在IsoP诱导的心肌保护中发挥关键作用。免疫印迹分析表明，与Ad-RFP感染的细胞组相比，Ad-CA-ALDH2-RFP感染的细胞组中的ALDH2水平大幅度增加（图2A）。持续活化的ALDH2显著抑制H/R引起的TUNEL阳性染色的增加，Caspase-3活性的增加和LDH的释放（P﹤0.05，图2B-E，）。而在Ad-CA-ALDH2介导ALDH2持续活化的情况下，再用IsoP则不能进一步增强上述心脏保护作用,即IsoP与未处理组无差异。这说明了活化的ALDH2（即磷酸化的ALDH2）参与IsoP诱导的心脏保护作用。
研究结论：IsoP通过激活AlDH2缓解了体外H/R损伤诱导的心肌细胞凋亡。ALDH2基因下调,H/R后心肌细胞凋亡数目大大增加，持续活化的ALDH2（即ALDH2基因上调）显著抑制H/R引起的心肌细胞凋亡，这表明活化的ALDH2参与了IsoP诱导的心肌保护作用。这一结果支持我们的假设即磷酸化ALDH2可能在IsoP诱导的心肌保护中发挥关键作用。ALDH2的抗心肌细胞凋亡作用的机制可能与其抑制Caspase3的激活从而抑制凋亡通路的激活有关。
研究目的：通过ALDH2激动剂及抑制剂，在药理水平激动或者抑制ALDH2，研究内源性ALDH2及其特异位点磷酸化在IsoP抵抗在体MIRI中的作用。
研究方法：采用结扎冠状动脉左前降支(LAD)40分钟之后进行120分钟再灌注处理制备雄性SD大鼠心肌I/R模型。为了证实异氟烷诱导的ＡＰＣ作用，从大鼠稳定期开始，以最小肺泡浓度1.0（2.1%）的异氟烷开始持续吸入30分钟，之后进行30分钟洗脱，再进行冠状动脉闭塞处理。根据不同的处理方法将大鼠随机分配到下列各组：假手术对照组和IsoP的假手术对照组：无/有IsoP，开胸分离冠状动脉左前降支，仅在LAD下穿线，但不阻断血流,持续160分钟(每组5只)；缺血再灌注组和IsoP的缺血再灌注组：无/有IsoP，可逆性的结扎LAD造成区域性心肌缺血40分钟，再灌注120分钟(每组5只)；为了评估ALDH2磷酸化在异氟烷诱导的心脏保护中的作用，在IsoP和无IsoP缺血处理前5分钟分别给予ALDH2的激活剂Alda-44（40μM）（每组8只），和ALDH2抑制剂cyanamide（5mM），（每组8只）。再灌注结束后，取出心脏采用伊文思蓝和TTC双重染色法测定心肌梗死面积。取出心脏前采集5ml血样，LDH和CK-MB的浓度用全自动生化分析仪7600及商用试剂盒进行测定；采用免疫印迹法测定信号通路蛋白(磷酸化的ALDH2and总ALDH2)的表达。
研究结果：(1)IsoP诱导的心脏保护作用：与假手术对照组相比，结扎LAD引起区域性心肌缺血40分钟然后进行120分钟再灌注使I/R组大鼠的血清LDH和CK-MB浓度和心梗面积显著增加(P<0.01)。而IsoP诱导的APC作用显著地降低了I/R所导致的心肌细胞LDH和CK-MB释放且大幅度地减少了I/R-诱导的心肌梗死面积（P<0.05）见图1；(2)ALDH2的磷酸化参与IsoP诱导的心脏保护:与I/R对照组相比，IsoP后使心肌细胞中磷酸化的ALDH2浓度显著升高（P<0.01），即缺血前IsoP增加了ALDH2的磷酸化。而ALDH2酶抑制剂cyanamide显著抑制了异氟烷诱导的ALDH2磷酸化后的激活效应（P<0.05）。使用ALDH2的直接激活剂Alda-44大幅度地增加了心肌细胞中ALDH2的磷酸化水平（P<0.01），但Alda-44并没有使IsoP诱导的ALDH2磷酸化效应进一步增强（图2A）。与ALDH2磷酸化相一致，我们观察到IsoP和使用Alda-44均显著减少了I/R引起的心肌细胞LDH和CK-MB向血浆的泄漏并减少了心梗面积（P<0.01，图2B-D）。IsoP和同时使用Alda-44不能进一步增强这种降低，即二者无叠加效应。然而，IsoP诱导的减少LDH和CK-MB释放和减少了心梗面积效应可明显被ALDH2抑制剂氰胺所阻断（P<0.01，图2B-D）。这些研究结果表明IsoP介导的心脏保护主要是由激活ALDH2所引起。
研究结论：IsoP诱导的APC作用显著减少了大鼠心脏I/R损伤所致的LDH和CK-MB释放，通过增加ALDH2的磷酸化减少了I/R-诱导的心肌梗死面积。我们推测IsoP介导的心脏保护可能主要是由激活ALDH2所引起。
研究目的：通过WesternBlot检测PKC两种亚型（PKCε和PKCδ）的线粒体转位，确定PKC两种亚型在IsoP抗MIRI中的活化情况；通过加入PKC两种亚型的抑制剂，研究PKC双亚型在ALDH2介导的IsoP抗MIRI中的作用。从而探索PKC信号通路与ALDH2分子的内源性信号转导途径与分子调控机制。
研究方法：(1)为了验证PKCε参与ALDH2的磷酸化，在IsoP和无IsoP缺血处理前5分钟给予雄性SD大鼠PKCε抑制剂PKCεV1-2（1μM）（每组8只），分别测定各实验组（I/R组、I/R+IsoP组、I/R+εV1-2组和I/R+IsoP+εV1-2组）大鼠的血清LDH和CK-MB的浓度，并用伊文思蓝-TTC双染色法测定心梗面积，并计算出心梗面积的百分比。Westernblot测定信号通路蛋白（Phos-ALDH2和Total-ALDH2、Mito-PKCε与Total-PKCε）的表达；(2)为了探讨IsoP对PKCδ的活化及向线粒体转位的影响，分别测定各实验组（SHAM组、I/R组、SHAM+IsoP组和I/R+IsoP组）的Mito-PKCδ和Total-PKCδ的表达量。（3）为了探讨PKCδ在ALDH2磷酸化中的作用，在IsoP和无IsoP缺血处理前5分钟给予PKCδ的抑制剂Rottlerin（1μM）（每组8只）。分别测定各实验组（I/R组、I/R+IsoP组、I/R+rottlerin组和I/R+IsoP+rottlerin组）大鼠血清中LDH和CK-MB的浓度。Westernblot测定信号通路蛋白（Phos-ALDH2和Total-ALDH2）的表达。研究结果：(1)PKCε参与了IsoP诱导的ALDH2的磷酸化和心脏保护：PKCε转位到线粒体后使ALDH2磷酸化是保护心脏免受I/R损伤的必需步骤。与未经
IsoP的I/R对照组相比，IsoP后心肌细胞线粒体中的PKCε浓度显著升高（P<0.01）同时伴有心肌细胞中磷酸化的ALDH2浓度显著升高（P<0.01），而PKCε抑制剂εV1-2能显著抑制这种升高（P<0.01）。在此，我们证明：IsoP导致PKCε在线粒体中浓度升高且伴随着ALDH2的磷酸化。IsoP介导的ALDH2的磷酸化被PKCε抑制剂εV1-2所抑制。由于PKCε在线粒体中的转位迅速发生，导致细胞质基质PKCε水平相应下降，因为细胞总的PKCε水平没有发生变化（图1A），因此我们的数据表明IsoP使PKCε动态转位到线粒体上以应对I/R引起的损伤。与PKCε转运到线粒体的情形相对应，PKCε抑制剂εV1-2取消了IsoP诱导的减少LDH和CK-MB释放效应以及减少心肌梗死面积的效应(图1B-D)；(2)IsoP减少了心肌细胞的PKCδ的活化及向线粒体转位：我们的数据表明：与假手术对照组相比，I/R导致心肌细胞线粒体中的PKCδ浓度显著增高（P﹤0.01），IsoP后大大减少了心肌细胞中的PKCδ向线粒体转位使心肌细胞线粒体中的PKCδ浓度显著降低（P﹤0.01）（图2A-B）（3）PKCδ向线粒体转位减少参与了IsoP诱导的心脏保护：IsoP减少了PKCδ从胞浆转位到线粒体中，PKCδ抑制剂rottlerin显著抑制了I/R导致的心肌细胞ALDH2磷酸化水平的降低（图3A-B）以及LDH和CK-MB释放（图3C-D），其效果与IsoP相当免疫印迹分析结果表明：使用PKCδ抑制剂rottlerin抑制了PKCδ的活性后,无论是否施用IsoP，I/R损伤后ALDH2磷酸化水平都显著升高。类似地，IsoP使PKCδ的活性被抑制从而使PKCδ线粒体移位减少以应对I/R引起的心肌损伤。研究结论：IsoP导致PKCε在线粒体中的浓度显著升高且伴随着ALDH2的磷
酸化，同时减少PKCδ的线粒体移位，通过双向调节PKC双亚型（PKCε和PKCδ）通路在心肌I/R过程中的活化水平，从而进一步激活了ALDH2，抵抗了心肌I/R损伤。即IsoP诱导的抗心肌I/R损伤机制可能是通过激活PKC（PKCε和PKCδ）-ALDH2信号通路实现的。
Objective:Confirm the critical role of ALDH2in isoflurane-induced cardioprotection and detect the functional significance of manipulating ALDH2expression by construction of ALDH2knockdown adenovirus and constitutively active ALDH2mutant adenovirus.
Methods (1) according to the sequence of ALDH2and the principle of SiRNA,we designed a siRNA refer to Cortinez, G's paper, target the ALDH2gene we synthesis small hairpin siRNA, after denature and refolding,formed double strand ALDH2-siRNA.Using the DNA recombination technology,we linked ALDH2-siRNA and lined RetroQ vector. After Identification by restriction endonuclease digestion and replication, we cut the Rat-ALDH2-SiRNA from RetroQ-Rat-ALDH2and link it to pShuttle-Basic-ERFP shuttle vector, get the pShuttle-RFP-Rat-ALDH2-siRNA vector. Finally we transfer pShuttle-RFP-Rat-ALDH2-siRNA to pAdeno vector to get pAdeno-RFP-Rat-ALDH2-siRNA vector.(2) Previous studies demonstrated that the constitutively active ALDH2amino acid487must be Glu not Lys, and Thr185, Thr412and Ser279must be constitutively phosphorylated. Accordingly, we obtained constitutively active mutant ALDH2(CA-ALDH2) by nucleotide substitutions leading to the mutations Lys487Glu, Thr185Asp, Thr412Asp, and Ser279Asp introduced into the wild-type rat ALDH2cDNA.Using the DNA recombination technology, We linked CA-ALDH to pShuttle-CMV-RFP shuttle vector,the pShuttlet-CA-ALDH2-RFP vector. We transfer pShuttle-CA-ALDH2-RFP to pAdeno vector to get pAdeno-CA-ALDH2-RFP vector.
Results:(1)Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector, after Identification by restriction endonuclease digestion, there is no mutation in the sequence, which is in our expectation.(2) Using the site-mutation technology we constructed pAdeno-CA-ALDH2vector after Identification by restriction endonuclease digestion, and got the right sequence, which Laid the foundation for our next experiments.
Conclusions:Successfully constructed pAdeno-RFP-Rat-ALDH2-siRNA vector,Western blot analysis showed siRNA can inhibite ALDH2gene expression in rat Myocardial cells;Successfully constructed pAdeno-CA-ALDH2vector,Western blot analysis showed the CA-ALDH2can expressed in Myocardial cells quite well.
Objective:Elucidate the role of ALDH2and in anti-apoptosis effect induced by isoflurane pretreatment by implementing specific activation or suppression on ALDH2gene, where neonatal rat cardiomyocytes were infected by pre-prepared ALDH2knockdown and constitutively active mutant adenovirus vectors.
Methods:primary cultured one-day old neonatal SD rat cardiomyocytes were randomly and homogeneously divided into different experimental groups as follows: vector-infected with and without isoflurane, Adeasy-Si-ALDH2-treated with and without isoflurane; vector-infected with and without isoflurane, Adeasy-CA-ALDH2-treated with and without isoflurane. Exposure to isoflurane was performed by incubating the cells for5minutes in0.5mM isoflurane (approximately1.0minimum alveolar concentration) in0.5%FBS DMEM. The isoflurane-containing medium was removed immediately before the onset of hypoxic conditions and the cells were washed with phosphate-buffered saline(PBS). Simulated I/R was achieved by culturing the Isolated neonatal cardiomyocytes in0.5%FBS DMEM in a hypoxia chamber, saturated with5%CO2/95%N2at37℃for24h and following reoxygenation for12h using0.5%FBS DMEM in the normal incubating condition. At the end of H/R, the apoptosis pathway proteins(Cleaved caspase-3and full-length caspase-3) in different cell groups were determined by Western Blot analysis; the caspase-3enzyme activity of cell lysate in different groups was detected by Caspase-3Colorimetric assay kit; apoptosis index was performed using terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling (TUNEL) with an in situ cell death detection kit, according to the manufacturer's protocol for cultured cells and the lactate dehydrogenase(LDH)activity released from the cytosol of damaged cells into the culture medium was measured by Cytotoxicity Detection Kit (LDH, Roche). The role of ALDH2downregulation or activation in isoflurane-induced myocardial anti-apoptotic effect was evaluated by the values above.
Results:Isoflurane preconditioning attenuated cardiomyocytes apoptosis induced by H/R injury in vitro:After24h of hypoxia followed by12h of reoxygenation we observed significant cardiomyocyte apoptosis demonstrated by increased DNA fragmentation using TUNEL staining, by laser scanning cytometry and caspase3activity in the vector control(Ad-RPF) group (Figure1B-E). Pretreatment with isoflurane significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure1B-E). However, when ALDH2was downregulated (Figure1A) in cardiomyocytes by Ad-Si-ALDH2, increased TUNEL positive staining level, more intense cleaved caspase-3staining, caspase3activity and LDH release (P>0.05, FigurelB-E) were observed, which supports the hypothesis that ALDH2might play a critical role in isoflurane-induced cardioprotection. Immunoblotting analysis showed a substantial increase in ALDH2level in Ad-CA-ALDH2-RFP-infected cells compared to Ad-RFP (Figure2A). Constitutively active ALDH2induced by Ad-CA-ALDH2significantly inhibited the H/R-induced increase in TUNEL positive staining, caspase3activity and leakage of LDH (P<0.05, Figure2B-E), which were not further increased by isoflurane treatment, i.e. no significant difference existed between control and isoflurane-pretreatment group. This suggests that activated (or phosphorylated) ALDH2involves isoflurane-induced cardioprotection.
Conclusions:Isoflurane preconditioning alleviated in vitro H/R injury by activation of ALDH2. Downregulated ALDH2gene leads to a great increase in apoptotic cardiomyocytes. Constitutively activated ALDH2(upregulated ALDH2gene) results in a significant inhibition of H/R induced cardiomyocytes apoptosis, which indicates that activated ALDH2involves isoflurane-induced cardioprotection. This result supports our hypothesis that phosphorylated ALDH2may play a critical role in isoflurane-induced cardioprotection. The anti-apoptotic mechanism of ALDH2may involves inhibition of caspase-3and hence inhibition of apoptotic pathway.
Objective:Reveal the role and mechanism of endogenic ALDH2and its phosphorylation at specific site in anti-apoptosis effect induced by isoflurane pretreatment by means of pharmacologic activation or inhibition of ALDH2in vivo.
Methods:The acute myocardial I/R injury model was performed by LAD ligation. To confirm isoflurane-induced APC, a minimal alveolar concentration of isoflurane of1.0(2.1%) was started at the end of the stabilization period and administered for30minutes, followed by30minutes of washout before coronary occlusion. Rats were randomly assigned to one of the following groups subjected to different protocols:Sham groups, non-ischemic control group of sham-operated rats without and with isoflurane:rats were opened chest without ligating LAD for160minutes (n=5,respectively); I/R group andIsoP+I/R group:40minutes of myocardial ischemia and120minutes of reperfusion without and with isoflurane (n=5,respectively). To evaluate the role of ALDH2in isoflurane-induced cardioprotection, the direct activator of ALDH2, Alda-44(40μM), was given5minutes prior to ischemia in the groups without and with isoflurane (n=8, respectively), and the ALDH2inhibitor, cyanamide (5mM), was given without and with isoflurane (5minutes prior to isoflurane)(n=8, respectively). At the end of the reperfusion, the heart was removed and infarct size(IS) and area at risk(AAR) were assayed by Evans Blue and TTC staining. Before removing the heart,5ml blood samples were taken. Lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) were assayed using commercial kits by an automatic analyzer7600; signal pathway proteins (Phos-ALDH2and Total-ALDH2in different groups) were assayed by Western Blot analysis.
Results:(1) Cardioprotective effect induced by Isoflurane preconditioning: Regional myocardial ischemia for40minutes by LAD ligation followed by120minutes of reperfusion markedly increased the leakage of LDH, CK-MB and myocardial infarctsize compared to sham controls(P<0.01). Isoflurane-induced APC significantly reduced the I/R-induced increase in LDH, CK-MB release in rat heart and substantially decreased I/R-induced myocardial infarct size (P<0.05). As shown in Figure1.(2) Phosphorylation of ALDH2participated in cardioprotection induced by IsoP:Pretreatment with isoflurane prior to ischemia increased the phosphorylation of ALDH2compared to I/R groups (P<0.01). The ALDH2inhibitor, cyanamide, significantly inhibited isoflurane-induced activation of ALDH2(P<0.05). The direct activator of ALDH2, Alda-44, substantially increased the phosphorylation of ALDH2P<0.01), but did not enhance the phosphorylation of ALDH2by isoflurane (图2A) Consistent with ALDH2phosphorylation,we observed that isoflurane and Alda-44markedly attenuated I/R-induced leakage of LDH and CK-MB in plasma, as well as myocardial infarct size (P<0.01,图2B-D) However, the isoflurane-induced decrease in LDH and CK-MB release, and reduction of infarct size was significantly blocked by the ALDH2inhibitor cyanamide (P<0.01, Figure2B-D). These findings suggest that isoflurane-mediated cardioprotection is mainly mediated by activation of ALDH2.
Conclusions:LDH and CK-MB leakage from rat heart I/R injury is significantly reduced by APC effect by isoflurane preconditioning, with a decrease in infarct size caused by upregulation of ALDH2phosphorylation. We suggest isoflurane-induced cardioprotective effect may essentially result from ALDH2activation.
Objective:Measure the activity of two PKC subtypes (PKCε and PKCδ) in isoflurane pretreatment against myocardial I/R injury by detection of mitochondrial translocation of both subtypes with Western blotting. Identify the role of both subtypes in ALDH2-mediated isoflurane pretreatment by application of inhibitors of both subtypes. Explore endogenic PKC and ALDH2signaling pathway and its molecular regulation mechanism.
Methods:(1) To verify that PKCε participates in the phosphorylation of ALDH2, the PKCε inhibitor, PKCε v1-2(1μM), was given5minites prior to ischemia without and with isoflurane (n=8, respectively,).Analyze the concentration of LDH and CK-MB in the rat Serum. Evans Blue-TTC stainning to analyze infarct area in different groups (I/R, I/R+IsoP, I/R+εvl-2and I/R+IsoP+εvl-2group). Signaling pathway proteins(Phos-ALDH2, Total-ALDH2, Mito-PKCε and Total-PKCε in Four different groups) were assayed by Western Blot analysis.(2) To identify the effect of isoflurane preconditioning on PKCδ activation and translocation, we measured mito-PKCδ and total PKCδ expression in all groups (Sham, I/R, Sham+IsoP and I/R+IsoP group).(3) To identify the role PKCδ plays in phosphorylation of ALDH2, the PKCδ inhibitor, Rottlerin(1μM), was given5minites prior to ischemia without and with isoflurane (n=8).Analyze the concentration of LDH and CK-MB in the Serum. Signaling pathway proteins(Phos-ALDH2, Total-ALDH2in Four different groups) were assayed by Western Blot analysis.
Results:(1) PKCε is involved in isoflurane-induced phosphorylation of ALDH2and cardioprotection:PKCε translocation to mitochondria and then phosphorylation of ALDH2is required to protect the heart from I/R injury. Compared with control group without isoflurane preconditioning, isoflurane pretreatment significantly enhanced mitochondrial PKCε concentration in cardiomyocytes (P<0.01), accompanied with significant increase in phosphorylated ALDH2in cardiomyocytes (P<0.01), whileεV1-2, a PKCε inhibitor, could significantly inhibit this effect (P<0.01). Here we demonstrate that pretreatment with isoflurane resulted in elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2. Isoflurane-induced phosphorylation of ALDH2was inhibited by the PKCε inhibitor, PKCεV1-2. Because mitochondrial translocation of PKCε occurs rapidly, with a corresponding decline in cytosolic PKCε levels, and because the total cellular PKCε levels do not change (FigurelA), our data suggest that pretreatment with isoflurane enables dynamic mitochondrial translocation of PKCε in response to I/R. Consistent with PKCε translocation to mitochondria, PKCεV1-2had a detrimental effect on isoflurane-induced attenuation of LDH and CK-MB leakage and the decrease in myocardial infarct size (Figure1B-D);(2) Pretreatment with isoflurane resulted in decreased PKCδ activity and mitochondrial levels in cardiomyocytes. Our data shows that compared to sham groups, I/R led to a significant increase in PKCδ levels in mitochondria of cardiomyocytes (P<0.01), Isoflurane preconditioning greatly inhibits mitochondria translocation of PKCδ in cardiomyocytes, and significantly decreases mitochondria PKCδ concentration in cardiomyocytes (P<0.01)(Figure2A-B).(3) Decreased level of PKCδ translocation to mitochondria involves isoflurane preconditioning-induced cardioprotection:Isoflurane-afforded inhibition of ALDH2phosphorylation level decrease (Figure3A-B) and LDH and CK-MB release (Figure3C-D) by I/R was mimicked by Rottlerin. Western blot showed that inhibition of PKCδ significantly elevated phosphorylation of ALDH2regardless of isoflurane preconditioning. Similarly, isoflurane preconditioning inhibits PKCδ activity, thus inhibiting its mitochondria translocation to act against I/R-induced myocardial injury.
Conclusions:Pretreatment with isoflurane resulted in significantly elevated mitochondrial levels of PKCε accompanied by phosphorylation of ALDH2, in the same time attenuated translocation of PKCδ. The bi-directional regulation of PKC two subtypes activation level during I/R would further activate ALDH2, and guarantee resistance to ischemia-reperfusion injury. Isoflurane preconditioning may activate PKC (PKCεand PKCδ)-ALDH2signaling pathway to induce protective effect against myocardial I/R injury.
引文
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