线粒体乙醛脱氢酶2(ALDH2)通过AMPK/FOXO3a通路改善高糖导致的心肌细胞凋亡

目的:观察乙醛脱氢酶2(ALDH2)对高糖诱导的H9C2心肌细胞存活及凋亡的影响,并探讨腺苷酸活化蛋白激酶(AMPK)/FOXO3a信号通路在高糖导致的心肌细胞凋亡中的调控作用。方法:以30 mmol/L葡萄糖诱导培养H9C2心肌细胞48 h,经ALDH2激动剂Alda-1及AMPK抑制剂Compound C干预后,用MTT法检测细胞的存活情况,TUNEL试剂盒检测细胞凋亡情况,Western blot检测ALDH2、磷酸化AMPK和FOXO3a蛋白的表达水平。结果:与对照组相比,高浓度葡萄糖培养H9C2心肌细胞后,细胞的存活率显著降低、凋亡指数明显升高,磷酸化AMPK的表达水平明显上调,ALDH2和磷酸化FOXO3a的蛋白表达显著降低(P0.05)。ALDH2的激动剂Alda-1处理可显著提高高糖诱导的H9C2心肌细胞的存活率、降低其凋亡率,减少磷酸化AMPK的蛋白表达,增加ALDH2的表达和FOXO3a蛋白的磷酸化;而进一步采用AMPK的抑制剂Compound C处理,可显著抑制Alda-1对高糖诱导的H9C2心肌细胞的这些影响。结论:ALDH2的激动剂Alda-1对高糖诱导的心肌细胞凋亡具有保护作用,可能与其激活AMPK,进而抑制心肌细胞FOXO3a的活性有关。     

虎杖苷对急性心肌梗死所致心脏损伤的保护作用

为了考察虎杖苷对急性心肌梗死所致心脏损伤的保护作用,本研究对H9c2大鼠心肌细胞进行缺氧诱导来模拟急性心肌梗死中心肌细胞的变化。然后用200μmol/L的虎杖苷处理心肌细胞12 h。考察虎杖苷对心肌细胞活力、细胞凋亡及相关蛋白(caspase-3, Bcl-2)和ROS生成的应用,并用小干扰RNA敲低Nrf2,考察敲低Nrf2对心肌细胞的影响。研究显示,缺氧处理可显著降低心肌细胞活力并增加细胞凋亡率,而虎杖苷可抑制缺氧诱导的细胞活力降低和细胞凋亡。虎杖苷可显著抑制缺氧诱导的caspase-3的下调并抑制缺氧诱导的Bcl-2的上调。虎杖苷可显著抑制缺氧诱导的Nrf2和HO-1的下调。敲低Nrf2可降低H9c2心肌细胞活力并增加细胞凋亡率。敲低Nrf2可上调caspase-3表达,并下调Bcl-2和Nrf2/HO-1信号通路的表达。缺氧可诱导H9c2细胞中ROS的产量升高,虎杖苷可抑制ROS的生成。然而,敲低Nrf2可导致细胞中ROS产量再次升高。虎杖苷具有抑制缺氧诱导的心肌细胞凋亡的作用,并且虎杖苷可通过抗氧化作用来减轻急性心肌梗死所致的心脏损伤。虎杖苷的抗氧化和心肌保护作用部分依赖于Nrf2/HO-1信号通路。     

喜树碱诱导甜菜夜蛾细胞凋亡线粒体途径的MPTP依赖性

【目的】线粒体通透性转换孔(MPTP)的开放可以导致线粒体膜通透性改变,与细胞凋亡关系密切。本研究旨在探索MPTP在喜树碱诱导的昆虫细胞凋亡中的作用,以进一步揭示喜树碱(CPT)诱导昆虫细胞凋亡的机制。【方法】环孢菌素A(CsA)为MPTP开放抑制剂,通过预加入20μmol/L CsA,应用流式细胞仪测定其对CPT和羟基喜树碱(HCPT)诱导的甜菜夜蛾Spodoptera exigua细胞(IOZCAS-SPEX-Ⅱ)凋亡作用的影响,包括细胞内Ca~(2+)浓度变化,线粒体膜电位变化以及活性氧簇(ROS)变化,从而分析MPTP在CPT和HCPT诱导细胞凋亡的作用。【结果】结果显示,10μmol/LCPT和HCPT处理IOZCAS-SPEX-Ⅱ细胞6 h和12 h时,与0.1%DMSO对照组相比,甜菜夜蛾细胞发生凋亡,胞质Ca~(2+)浓度增大,线粒体膜电位降低或丧失,ROS增加,即CPT和HCPT诱导甜菜夜蛾细胞发生凋亡,为线粒体内途径。但经过20μmol/L CsA预处理2 h后再加入CPT和HCPT处理6 h,与0.1%DMSO组相比,细胞凋亡率、胞质Ca~(2+)浓度、线粒体膜电位及ROS产生均无显著差异(P0.05),即CsA抑制了MPTP的开放,从而抑制了CPT和HCPT诱导的甜菜夜蛾细胞凋亡;而加入CPT和HCPT处理12 h时,CsA对MPTP开放的抑制作用显著降低,与单CPT和HCPT处理组相比,细胞凋亡率、胞质Ca~(2+)浓度、线粒体膜电位及ROS差异不显著,即CPT和HCPT诱导的细胞凋亡如常发生。【结论】本研究证实喜树碱和羟基喜树碱诱导甜菜夜蛾细胞凋亡线粒体途径具有MPTP开放依赖性,且首次明确这种依赖性具有时间性。     

新狼毒素A诱导人黑色素瘤A375细胞凋亡的机制研究

为探讨新狼毒素A抑制人黑色素瘤A375细胞增殖及诱导凋亡的作用。本实验采用噻唑蓝(MTT)法检测新狼毒素A对人黑色素瘤A375细胞增殖的影响,Hoechst33258法观察细胞形态,流式细胞仪检测细胞凋亡率和线粒体膜电位,Westernblot检测凋亡相关蛋白表达。结果显示新狼毒素A以时间剂量依赖性的方式显著抑制A375细胞的增殖;不同浓度新狼毒素A(0、15、30、45μmol/L)作用于A375细胞48h后细胞出现显著凋亡特征,新狼毒素A增加A375细胞的凋亡率且降低了线粒体膜电位,上调Bax、caspase-3和细胞色素C(CytochromeC)蛋白的表达,下调Bcl-2蛋白的表达。以上结果说明新狼毒素A抑制A375细胞增殖,诱导细胞凋亡,其机制可能与诱导线粒体途径的凋亡有关。     

曲古霉素A诱导人膀胱癌细胞周期阻滞和凋亡的研究

目的:探讨组蛋白去乙酰化酶抑制剂曲古霉素A(TSA)对人膀胱癌T24细胞周期和凋亡的影响。方法:以不同剂量TSA(0.1μM,0.3μM和1μM)处理T24细胞。采用MTT法检测细胞存活率,AnnexinV-PI染色检测细胞凋亡,流式细胞仪检测caspase-3活性,Western blot法检测P21蛋白表达。结果:TSA剂量依赖性降低膀胱癌细胞存活率,促进细胞凋亡,表现为AnnexinV阳性细胞明显增多,同时活化的caspase-3水平增高。TSA还可通过诱导膀胱癌细胞周期阻滞于G2/M期抑制细胞生长,且呈剂量依赖性。结论:TSA通过促进caspase-3激活诱导膀胱癌细胞凋亡,同时诱导细胞阻滞于G2/M期。     

柯里拉京对人肺癌细胞A549的抑制作用及其机制研究

该研究旨在探讨柯里拉京对人肺癌A549细胞凋亡的影响及其潜在作用机制。采用CCK-8细胞活性检测试剂盒检测柯里拉京对A549细胞活性的影响;通过流式细胞术检测细胞凋亡;JC-1线粒体膜电位检测试剂盒检测线粒体膜电位;免疫印迹法检测凋亡相关蛋白(bax、bcl-2、cleaved-caspase-3、cleaved-PARP)的表达量;通过DCFH-DA探针标记检测细胞内ROS水平。研究结果显示,柯里拉京处理能够剂量依赖性地抑制A549细胞的活性,并通过上调bax的表达、下调bcl-2的表达,破坏线粒体膜电位,促进有活性的cleaved-caspase-3以及cleaved-PARP的形成,诱导A549细胞凋亡。活性氧清除剂NAC能够明显逆转柯里拉京诱导的细胞凋亡。因此,柯里拉京可能通过调节胞内ROS水平诱导人肺癌细胞A549发生凋亡。     

细胞周期蛋白依赖激酶Roscovitine 诱导非小细胞肺癌A549 细胞凋亡及 其机制的研究

目的:探讨细胞周期蛋白依赖激酶(CDK)抑制剂Roscovitine(Ros)诱导非小细胞肺癌(NSCLC)A549细胞凋亡及其作用机制。方法:以不同浓度Ros(10μM、20μM、40μM)处理细胞24h,采用Annexin V-PI染色以流式细胞仪检测细胞凋亡,Westernblot法检测胞浆中和线粒体促凋亡蛋白Bax和Bad的表达,流式细胞仪检测线粒体膜电位(MMP)变化。结果:Ros以剂量依赖的方式诱导A549细胞凋亡,同时Bad和Bax在胞浆的含量随着Ros剂量的增加而减少,而在线粒体中却出现相反的结果,线粒体膜电位随Ros剂量的增大而降低。结论:Ros可通过促进Bax和Bad由胞浆向线粒体易位,诱导NSCLC A549细胞由线粒体途径发生凋亡。     

ROS在镉诱导HK-2细胞氧化损伤和凋亡中的作用研究

该文研究了活性氧(reactive oxygen species, ROS)在镉(Cadmium, Cd)诱导HK-2细胞氧化损伤和凋亡中的作用。不同浓度CdCl2处理HK-2细胞不同时间后,通过MTT法、DCFH-DA标记、JC-1染色、彗星实验和流式细胞术分别检测细胞活性、ROS、线粒体膜电位Δψm、DNA损伤及细胞凋亡情况。结果显示, CdCl2处理引起HK-2细胞形态皱缩、变圆,活性下降,且呈时间和剂量依赖性; CdCl2处理导致ROS水平升高、线粒体膜电位Δψm下降、DNA损伤和caspase-3活化,最终导致细胞凋亡,且60μmol/L处理组及高浓度组与对照组相比差异极显著(P0.01)。采用ROS清除剂NAC与CdCl2共处理细胞24 h,发现细胞形态明显恢复、ROS水平显著降低、线粒体膜电位Δψm显著升高、彗星尾部长度和DNA百分比显著下降、凋亡细胞减少(P0.01)。综上所述, ROS介导了Cd诱导的HK-2细胞氧化损伤和凋亡。     

黄芩苷干预甲型H1N1流感病毒感染诱导的A549细胞周期分布及凋亡

以甲型H1N1流感病毒作为刺激因素,在人肺腺癌A549细胞培养内采用MTT比色法和细胞病变(CPE)法观察黄芩苷不同作用时间的抗病毒效率;以碘化丙啶(Propidium iodide,PI)单染流式细胞仪分析细胞周期中各时期的细胞百分数和对细胞增殖的影响,以Hoechst33258染色荧光显微镜观察细胞凋亡的形态学变化,并采用免疫荧光实验测定膜受体通路Caspase 8和线粒体通路Caspase 9及作为细胞凋亡标志的Caspase 3的活性。结果显示:流感病毒感染36h后宿主细胞周期阻滞于S期(P<0.05),并在G0期细胞峰前出现一个亚二倍体细胞峰(凋亡细胞峰)。A549细胞中Caspase 8/3活性明显升高,但标记Caspase 9活性的荧光亮度增强不明显。黄芩苷对甲型流感病毒感染诱导的细胞损伤有较好的保护作用,最大剂量的黄芩苷100μg/mL无毒,可抑制病毒细胞病变的产生,50~100μg/mL治疗组可明显调节流感病毒感染诱导的细胞周期分布(P<0.05),细胞凋亡现象明显减少,100μg/mL黄芩苷治疗组Caspase 8/3活性显著降低,接近正常对照组细胞水平,有剂量依赖性。实验说明:黄芩苷可拮抗甲型流感病毒H1N1细胞病变,调控细胞周期分布,并通过抑制Caspase 8的激活,进一步抑制Caspase 3活性,从而发挥抗病毒作用。     

曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖和凋亡的作用及其机制

观察曲古抑菌素A（TSA）对骨肉瘤细胞株143B增殖及凋亡的作用,并探讨其机制。TSA与p38抑制剂（SB203580,3μmol／L）及JNK抑制剂（SP600125,0．5μmol／L）单独或同时处理143B细胞,分别以MTT、台盼蓝染色、流式细胞术和JC—1（测定线粒体跨膜电位）法检,TSA对143B细胞的增殖、存活、周期以及凋亡的影响。应用RT-PCR、Westernblot检测Bax、Bcl．2、p38／JNK表达。结果显示,TsA能够以时间和剂量依赖方式抑制143B细胞增殖,使细胞周期阻滞于GdG。与G2／M期,并能诱导143B细胞凋亡,引起线粒体膜电位降低,促凋亡蛋白Bax表达上调,抑凋亡蛋白Bcl．2表达下调,同时使p38／ⅢK活化增加。p38／JNK4检测剂则能逆转TSA对Bax／Bcl．2的上调及抑制作用。研究结果揭示,TSA可以时间剂量依赖方式抑制143BN胞增殖,阻滞细胞周期,诱导细胞凋亡;其诱导细胞凋亡的机制可能与活化MAPK通路中p38和JNK的活性从而激发线粒体凋亡通路有关。     

Increased expression of microRNA-378a-5p in acute ethanol exposure of rat cardiomyocytes

Alcohol abuse is a risk factor for a distinct form of congestive heart failure, known as alcoholic cardiomyopathy (ACM). Here, we investigate how microRNAs may participate in the induction of cardiomyocyte apoptosis associated with ethanol exposure in vitro. Increasing the concentrations of ethanol to primary rat cardiomyocytes resulted in elevated apoptosis assessed by annexin V and propidium iodide staining, and reduced expression of an enzyme for alcohol detoxification aldehyde dehydrogenase 2 (ALDH2). These ethanol effects were accompanied by a substantial elevation of miR-378a-5p. Driving miR-378a-5p overexpression in cardiomyocytes decreased ALDH2. The specific interaction of miR-378a-5p with the 3’UTR of ALDH2 was examined by luciferase reporter assays, and we found that miR-378a-5p activity depends on a complementary base pairing at the 3′-UTR region of ALDH2 mRNA. Finally, ethanol-induced apoptosis in cardiomyocytes was attenuated in the presence of anti-miR378a-5p. Collectively, these data implicate a likely involvement of miR-378a-5p in the stimulation of cardiomyocyte apoptosis through ALDH2 gene suppression, which might play a potential role in the pathogenesis of ACM.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

Ubiquitination and degradation of the anti-apoptotic protein ARC by MDM2

Current evidence shows that cardiomyocyte apoptosis plays a central role in the pathogenesis of myocardial disease and that reactive oxygen species is critically responsible for mediating cardiomyocyte apoptosis in both ischemia-reperfusion injury and dilated cardiomyopathy. ARC (Apoptosis Repressor with Caspase recruitment domain) is an anti-apoptotic protein that is found abundantly in terminally differentiated cells such as cardiomyocytes. The ARC knock-out mouse developed larger infarct in response to ischemia-reperfusion and transitioned more rapidly and severely to dilated cardiomyopathy following aortic constriction. In addition, ARC protein levels are decreased in human dilated cardiomyopathy and when cardiomyocytes are exposed to oxidative stress in vitro, but the mechanisms regulating ARC protein levels are not known. Here we show that degradation of ARC is dependent on the p53-induced ubiquitin E3 ligase, MDM2. Oxidative stress reduced ARC levels and up-regulated MDM2. MDM2 directly accelerated ARC protein turnover via ubiquitination and proteasomal-dependent degradation. This activity requires a functioning MDM2 ring finger domain because the MDM2(C464A) mutant was unable to direct ARC degradation. Furthermore, ARC degradation requires MDM2, because MDM2 knock-out fibroblasts showed defective ARC degradation that could be rescued by MDM2. Proteasomal inhibitors rescued both MDM2 and H(2)O(2)-induced degradation of ARC and inhibited cardiomyocyte apoptosis. Dilated cardiomyopathic hearts from mice that have undergone transverse aortic banding have increased MDM2 levels associated with decreased ARC levels. We conclude that MDM2 is a critical regulator of ARC levels in cardiomyocytes. Prevention of MDM2-induced degradation of ARC represents a potential therapeutic target to prevent cardiomyocyte apoptosis.     

Effects of short‐chain acyl‐CoA dehydrogenase on cardiomyocyte apoptosis

Short‐chain acyl‐CoA dehydrogenase (SCAD), a key enzyme of fatty acid β‐oxidation, plays an important role in cardiac hypertrophy. However, its effect on the cardiomyocyte apoptosis remains unknown. We aimed to determine the role of SCAD in tert‐butyl hydroperoxide (tBHP)‐induced cardiomyocyte apoptosis. The mRNA and protein expression of SCAD were significantly down‐regulated in the cardiomyocyte apoptosis model. Inhibition of SCAD with siRNA‐1186 significantly decreased SCAD expression, enzyme activity and ATP content, but obviously increased the content of free fatty acids. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as cardiomyocytes treated with tBHP, such as the increase in cell apoptotic rate, the activation of caspase3 and the decrease in the Bcl‐2/Bax ratio, which showed that SCAD may play an important role in primary cardiomyocyte apoptosis. The changes of phosphonate AMP‐activated protein kinase α (p‐AMPKα) and Peroxisome proliferator‐activated receptor α (PPARα) in cardiomyocyte apoptosis were consistent with that of SCAD. Furthermore, PPARα activator fenofibrate and AMPKα activator AICAR treatment significantly increased the expression of SCAD and inhibited cardiomyocyte apoptosis. In conclusion, for the first time our findings directly demonstrated that SCAD may be as a new target to prevent cardiomyocyte apoptosis through the AMPK/PPARα/SCAD signal pathways.     

Isoflurane Preconditioning Confers Cardioprotection by Activation of ALDH2

The volatile anesthetic, isoflurane, protects the heart from ischemia/reperfusion (I/R) injury. Aldehyde dehydrogenase 2 (ALDH2) is thought to be an endogenous mechanism against ischemia-reperfusion injury possibly through detoxification of toxic aldehydes. We investigated whether cardioprotection by isoflurane depends on activation of ALDH2.Anesthetized rats underwent 40 min of coronary artery occlusion followed by 120 min of reperfusion and were randomly assigned to the following groups: untreated controls, isoflurane preconditioning with and without an ALDH2 inhibitor, the direct activator of ALDH2 or a protein kinase C (PKCε) inhibitor. Pretreatment with isoflurane prior to ischemia reduced LDH and CK-MB levels and infarct size, while it increased phosphorylation of ALDH2, which could be blocked by the ALDH2 inhibitor, cyanamide. Isolated neonatal cardiomyocytes were treated with hypoxia followed by reoxygenation. Hypoxia/reoxygenation (H/R) increased cardiomyocyte apoptosis and injury which were attenuated by isoflurane and forced the activation of ALDH2. In contrast, the effect of isoflurane-induced protection was almost abolished by knockdown of ALDH2. Activation of ALDH2 and cardioprotection by isoflurane were substantially blocked by the PKCε inhibitor. Activation of ALDH2 by mitochondrial PKCε plays an important role in the cardioprotection of isoflurane in myocardium I/R injury.     

Deficiency in a mitochondrial aldehyde dehydrogenase increases vulnerability to oxidative stress in PC12 cells

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) plays a major role in acetaldehyde detoxification. The alcohol sensitivity is associated with a genetic deficiency of ALDH2. We have previously reported that this deficiency influences the risk for late-onset Alzheimer's disease. However, the biological effects of the deficiency on neuronal cells are poorly understood. Thus, we obtained ALDH2-deficient cell lines by introducing mouse mutant Aldh2 cDNA into PC12 cells. The mutant ALDH2 repressed mitochondrial ALDH activity in a dominant negative fashion, but not cytosolic activity. The resultant ALDH2-deficient transfectants were highly vulnerable to exogenous 4-hydroxy-2-nonenal, an aldehyde derivative generated by the reaction of superoxide with unsaturated fatty acid. In addition, the ALDH2-deficient transfectants were sensitive to oxidative insult induced by antimycin A, accompanied by an accumulation of proteins modified with 4-hydroxy-2-nonenal. Thus, these findings suggest that mitochondrial ALDH2 functions as a protector against oxidative stress.     

Inhibition of phospholipase C-gamma 1 augments the decrease in cardiomyocyte viability by H2O2

The present study was conducted to examine the role of a major cardiac phospholipase C (PLC) isozyme, PLC-gamma 1, in cardiomyocytes during oxidative stress. Left ventricular cardiomyocytes were isolated by collagenase digestion from adult male Sprague-Dawley rats (250-300 g) and treated with 20, 50, and 100 microM H2O2 for 15 min. A concentration-dependent (up to 50 microM) increase in the mRNA level and membrane protein content of PLC-gamma 1 was observed with H2O2 treatment. Furthermore, PLC-gamma 1 was activated in response to H2O2, as revealed by an increase in the phosphorylation of its tyrosine residues. There was a marked increase in the phosphorylation of the antiapoptotic protein Bcl-2 by H2O2; this change was attenuated by a PLC inhibitor, U-73122. Although both protein kinase C (PKC)-delta and -epsilon protein contents were increased in the cardiomyocyte membrane fraction in response to H2O2, PKC-epsilon activation, unlike PKC-delta, was attenuated by U-73122 (2 microM). Inhibition of PKC-epsilon with inhibitory peptide (0.1 microM) prevented Bcl-2 phosphorylation. Moreover, different concentrations (0.05, 0.1, and 0.2 microM) of this peptide augmented the decrease in cardiomyocyte viability in response to H2O2. In addition, a decrease in cardiomyocyte viability, as assessed by trypan blue exclusion, due to H2O2 was also seen when cells were pretreated with U-73122 and was as a result of increased apoptosis. It is therefore suggested that PLC-gamma 1 may play a role in cardiomyocyte survival during oxidative stress via PKC-epsilon and phosphorylation of Bcl-2.     

Interrelations between oxidative stress and calcineurin in the attenuation of cardiac apoptosis by eugenol

In view of the known involvement of oxidative stress and calcineurin (Ca²⁺-calmodulin dependent protein phosphatase) in β-Adrenergic stimulated events, we examined the influence of eugenol (an antioxidant generally regarded as safe by the Food and Agricultural Organization of the United Nations) on isoproterenol-induced apoptosis in neonatal cardiomyocytes. In comparison to unstimulated controls, cardiomyocytes stimulated with 50 μM isoproterenol for 48 h demonstrated (a) increased intracellular Ca²⁺ levels (b) oxidative stress involving enhanced reactive oxygen species, decreased GSH/GSSG ratio, enhanced lipid peroxidation, increased activities of superoxide dismutase and glutathione peroxidase (c) apoptosis, evidenced by increased number of annexin V/TUNEL positive cells, enhanced membrane fluidity, decreased mitochondrial membrane potential, increased activities of caspase 3 and 9 along with (d) increased calcineurin activity. Pre-incubation of cardiomyocytes with 100 μM eugenol for 1 h, followed by isoproterenol treatment for 48 h, led to reversal of enhanced intracellular Ca²⁺ levels, oxidative stress, calcineurin activation and apoptosis caused by isoproterenol. In addition, similar treatment of cardiomyocytes with 10 nM FK506, a calcineurin inhibitor, could also attenuate isoproterenol-induced apoptosis. These results indicate the beneficial effects of eugenol in preventing cardiomyocyte apoptosis.