【作者】 赵鹏；

【导师】 朱兴雷；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 第一部分Toll样受体4缺失改善小鼠心脏因缺血再灌注引起的功能不全与损伤背景与目的随着在临床缺血性心脏病的治疗中冠状动脉介入手术及冠状动脉搭桥手术的广泛普遍开展,由缺血再灌注引起的心脏损伤日益引起临床工作者的重视。在过去的几年中,除了药物及手术甚至基因等治疗手段的研究,一些动物基础实验及临床测定观察发现Toll样受体4（Toll-like receptor-4,TLR-4）在肝肾缺血再灌注的器官功能及细胞损伤病理中扮演重要角色,并且关于Toll样受体在各个器官中的作用是目前基础及临床研究及讨论的热点。然而心脏缺血再灌注损伤中Toll样受体4会有何作用及作用机制目前还不甚明确。冠心病是指冠状动脉粥样硬化使血管腔狭窄或阻塞,或（和）冠状动脉功能性改变（痉挛）导致心肌缺血缺氧或坏死而引起的心脏病。Toll样受体4是心脏及血管细胞膜上在先天免疫中革蓝氏阴性菌外膜组成成分脂多糖的近端受体。由缺血损伤引起的氧化应激造成脂多糖及热休克蛋白和纤连蛋白等配体可与Toll样受体4结合,并已在人类心脏心力衰竭和缺血中观察到Toll样受体4的表达增加。丝裂原激活的蛋白激酶（Mitogen activated protein kinase,MAPK）如p38丝裂原活化蛋白激酶（p38 Mitogen activated protein kinase,P38MAPK）,氨基末端激酶（c-Jun N-terminal kinase c-Jun,JNK）,胞外信号调节激酶（extracellularsignal-regulated kinase,ERK）是Toll样受体4的下游激活蛋白。先前的研究已发现缺血可激活p38丝裂原活化蛋白激酶,表现出心脏负性变力和限制性的舒张功能不全。腺苷酸活化蛋白激酶（AMP-activated Protein Kinase,AMPK）是一种重要的蛋白激酶,作为机体能量“监测器”,其活性受腺苷酸活化蛋白/三磷酸腺苷比例调控。已有的研究表明它对缺血引起的心脏损伤有积极的保护作用。腺苷酸活化蛋白激酶可被多种刺激因素激活,如运动,缺氧,缺血和应激等。它在调控细胞能量状态代谢稳定和控制能量产生和消耗中发挥主导作用。有趣的是已有报道说明它保护由缺氧引起的心脏损害的原因之一是因为减轻了内质网应激（endoplasmic reticulum stress,ER stress）。内质网应激是指由于某种原因导致细胞内质网内稳态失衡、生理功能发生紊乱的一种亚细胞器的病理过程。在真核细胞中,粗糙型内质网主要与蛋白质合成和初步修饰、加工（如蛋白质糖基化等）和转运及与膜之生成有关;光滑型内质网主要与糖原分解、脂类（包括磷脂和类固醇等）合成、细胞解毒作用及参与横纹肌收缩活动等有关。然而Toll样受体4在因缺血再灌注引起的心脏功能不全与损伤中会有何作用和其机制是目前还未阐明的问题。此研究的目的在于应用小鼠心脏缺血再灌注模型观察Toll样受体4缺失是否会对功能不全与损伤有保护作用,并进一步探讨保护作用机制是否通过调节腺苷酸活化蛋白激酶和丝裂原激活的蛋白激酶这两种重要调节酶并检测内质网应激相关蛋白。材料与方法野生型C3H/HeN和Toll样受体4（TLR-4）缺失C3H/HeJ小鼠购自美国Jackson Laboratory。C3H/HeJ小鼠因TLR-4的基因Tlr4^Lps-d发生突变,所以不表达功能性的TLR-4蛋白。小鼠麻醉后,在小动物呼吸机辅助呼吸下开胸后显微镜下结扎与开放左冠状动脉前降支制作缺血再灌注模型。开胸后15分钟缺血,然后60,120分钟再灌注。实验分为八组:（1）野生型C3H/HeN小鼠假手术组（n=7）,（2）野生型C3H/HeN小鼠缺血再灌注60分钟组（n=7）,（3）野生型C3H/HeN小鼠缺血再灌注120分钟组（n=7）,（4）野生型C3H/HeN小鼠缺血15分钟组（n=7）,（5）Toll样受体4（TLR-4）缺失C3H/HeJ小鼠假手术组（n=7）,（6）TLR-4缺失C3H/HeJ小鼠缺血再灌注60分钟组（n=7）,（7）TLR-4缺失C3H/HeJ小鼠缺血再灌注120分钟组（n=7）,（8）TLR-4缺失C3H/HeJ小鼠缺血15分钟组（n=7）。缺血再灌注后双染色技术测定心梗面积。Evan’s blue染色区域,TTC染色区域和TTC未染色区域的大小用Image Pro Plus软件测定。心肌细胞的凋亡使用caspase-3活性荧光测定法和TUNEL试剂盒。酶联免疫吸附测定法检测血清促炎症因子如肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）和白介素（interleukin,IL）1β和IL-6水平。丝裂原激活的蛋白激酶（Mitogenactivated protein kinase,MAPK）和腺苷酸活化蛋白激酶（AMP-activated ProteinKinase,AMPK）及其下游蛋白乙酰辅酶A羧化酶（Acetyl-CoA carboxylase,ACC）,真核生物翻译延伸因子-2（eukaryotic translation elongation factor-2,eEF2）和内质网应激标志蛋白生长抑制和DNA损伤诱导基因153（Growth arrest and DNAdamage induced gene 153,Gadd153）,糖调节蛋白78（glucose-regulated protein 78,GRP78）和肌醇内质网跨膜激酶（inositol-requiring and ER-to-nucleus enzyme,IRE）用免疫印迹方法来检测。数据均用均数±标准误表示。多组之间使用GraphPad Prism 4中方差分析（ANOVA）与Newman-Keuls post hoc检验。P值小于0.05认为有统计学意义。结果用双染色技术测定心肌梗死心肌坏死程度。结果显示在缺血再灌注后与野生型C3H/HeN小鼠相比,Toll样受体-4（TLR-4）缺失C3H/HeJ小鼠心脏梗死面积要小。缺血再灌注可促使心肌细胞发生凋亡,caspase-3活性与TUNEL阳性核在C3H/HeN组与C3H/HeJ组之间有明显差异,在C3H/HeN组较高。酶联免疫吸附测定法检测血清促炎症因子如TNF-α,IL-1β和IL-6水平。结果示小鼠心脏缺血再灌注后血清中TNF-α,IL-1β和IL-6在C3H/HeJ小鼠中较野生型有所减少。以上数据表明TLR-4缺失可以保护因缺血再灌注引起的损伤。为了进一步探讨Toll样受体-4缺失对缺血再灌注损伤保护的机制,我们用免疫印迹法检测心肌组织中腺苷酸活化蛋白激酶（AMPK）。结果显示与假手术组比较缺血及缺血再灌注均可使腺苷酸活化蛋白激酶的第172位的苏氨酸被磷酸化而激活,但在C3H/HeJ组中较C3H/HeN组有明显增高。同样的结果在腺苷酸活化蛋白激酶的下游蛋白如乙酰辅酶A羧化酶和真核生物翻译延伸因子-2可以发现。丝裂原激活的蛋白激酶（Mitogen activated protein kinase,MAPK）是Toll样受体4的下游激活蛋白,结果示与假手术比较p38丝裂原活化蛋白激酶（p38Mitogen activated protein kinase,P38MAPK）,氨基末端激酶（c-Jun N-terminalkinase c-Jun,JNK）和胞外信号调节激酶（extracellular signal-regulated kinase,ERK）在缺血及缺血再灌注均被激活,但是与炎症有关的p38和JNK在C3H/HeJ与C3H/HeN小鼠之间有统计学差异,C3H/HeJ小鼠明显低于C3H/HeN小鼠,有趣的是ERK却是相反的,C3H/HeJ小鼠明显高于C3H/HeN小鼠,可能是因为TLR-4缺失激活了与细胞生存活化相关的ERK。已有的研究报道缺血可以导致心肌细胞中各种原因导致的未折叠蛋白或错误折叠蛋白在内质网腔内的积聚,被称为内质网应激。由于内质网应激的发生,可诱导分子伴侣的产生和蛋白质翻译减少等。我们因此观察TLR-4缺失在缺血再灌注后内质网应激的变化。结果示缺血激活了内质网应激分子伴侣Gadd153,Grp78和IRE1α,而C3H/HeJ小鼠的激活水平明显低于C3H/HeN小鼠。结论1.Toll样受体-4缺失减小了缺血再灌注后心脏梗死的面积;2.Toll样受体-4缺失减少了缺血再灌注后心肌细胞的凋亡;3.小鼠心脏缺血再灌注后血清中促炎症因子TNF-α,IL-1β和IL-6在Toll样受体-4缺失鼠中较野生型有所减少;4.在缺血再灌注Toll样受体-4缺失小鼠中腺苷酸活化蛋白激酶（AMPK）的激活增强;5.在缺血再灌注Toll样受体-4缺失小鼠中其丝裂原激活的蛋白激酶（MAPK）的变化有不同;6.与野生型相比Toll样受体-4缺失减轻了因缺血再灌注引起的内质网应激。第二部分心脏特异性过表达胰岛素样生长因子1减轻由内毒素诱导的小鼠心功能不全和应激信号的激活背景与目的胰岛素样生长因子1（Insulin-Like Growth Factor I,IGF-1）在维持机体结构和功能方面起着重要作用。IGF-1参与许多生理及病理生理过程,如组织重构,葡萄糖与脂代谢和影响胰岛素敏感性等,在心脏方面对心肌细胞发育与心脏结构与重构发挥作用。IGF-1的缺乏会引起机体组成成分,细胞因子和神经内分泌因子活性的改变。已有的研究发现在心脏中IGF-1的不足会造成心脏萎缩与功能损害。严重的IGF-1缺乏的阿姆氏矮小鼠表现为寿命缩短和心肌细胞中兴奋收缩偶联的损害。在临床病人的研究观察中发现IGF-1缺乏同样会造成寿命缩短及心功能损害,表现为左室重量的减少,射血分数减低及舒张障碍。脂多糖（Lipopolysaccharide,LPS）是革蓝氏阴性菌外膜组成成分之一,对保持细菌完整结构有积极作用,它可以保护细菌免受外来某些对其有危害的化学物质的攻击。脂多糖还可以增强细胞膜负电位从而维持整个细菌胞膜的稳定。脂多糖是一种内毒素,可以引起动物机体强烈的免疫反应,它与多种细胞特别是巨噬细胞通过Toll样受体4（Toll-like receptor 4,TLR-4）结合,促使促炎症因子的分泌。LPS诱导模型作为急性模型可在短时间内使模型动物到达败血症晚期,而且方法简单有效。LPS模型对于研究败血症休克晚期的机体功能改变及其机制至今仍然是一个有效的手段。目前在美国严重败血症仍是住院病人主要死亡原因之一,这种死亡多由于因多器官衰竭造成的休克。在败血症病人及脂多糖诱导的实验性败血症中心功能损害是常见现象。在败血症病人血循环中胰岛素样生长因子1含量常低于常量。先前的研究表明心脏特异性过表达的IGF-1转基因小鼠能够降低心肌细胞凋亡,减轻心室壁的压力和抑制心梗后或随着年龄的增长心腔的扩大,在维持心脏形态和功能上有着至关重要的作用。有研究发现调节IGF-1的水平能够改善败血症患者的总体生存率,这可能是通过加强肝脏清除细菌毒素的作用和提高细胞免疫实现的。然而IGF-1在败血症中心脏损害作用机制还不甚明朗。本文就此应用脂多糖诱导的小鼠败血症模型研究胰岛素样生长因子1在心肌细胞收缩功能,胞内Ca²⁺处理功能,应激信号的作用及内质网应激中的作用机制。材料与方法年龄（16-20周）与体重（20-25克）配对的雄性FVB与心脏特异性过表达胰岛素样生长因子1（IGF-1）小鼠用于此实验。IGF-1转基因小鼠由纽约大学医学院Piero Anversa博士惠赠。实验分为四组:FVB小鼠对照组（以下文中简写为FVB）,FVB小鼠脂多糖处理组（FVB-LPS）,IGF-1转基因小鼠对照组（IGF-1）和IGF-1小鼠脂多糖处理组（IGF-1-LPS）。处理组小鼠腹腔注射在消毒生理盐水中溶解的Escherichia Coli脂多糖（Sigma,USA）根据已发表的文献选择剂量与时间,剂量为4 mg/kg,处理时间为6小时,6小时后处死小鼠,继续进行下列相关实验。根据试剂盒说明应用酶联免疫吸附测定法（ELISA）测定血清中IGF-1水平。在Langendorff系统应用Liberase Blendzyme 4酶（Sigma,USA）消化分离单个心肌细胞。单个心肌细胞的存活率为70%左右,选择外形边缘清楚收缩自如的单心肌细胞做为观察对象。将低于致死剂量的Escherichia Coli脂多糖（1μg/ml）与外源性重组IGF-1（50 nM）加入或不加入分离好的FVB单心肌细胞进行体外实验。使用SoftEdge MyoCam system（IonOptix Corporation,Milton,MA,USA）系统评估单个心肌细胞的收缩和舒张功能。An IonOptix SoftEdge software软件适用于捕获心肌细胞在收缩和舒张时长度的变化。细胞收缩和舒张通过下列指标进行评估:收缩峰值（peak shortening,PS）代表细胞收缩时缩短的幅度;最大收缩和舒张速率积分（±dL/dt）代表细胞收缩和舒张的最大速率;到达收缩峰值的时间（timeto PS,TPS）代表细胞收缩时程;90%舒张时间(time to 90%relengthening,TR₉₀)代表细胞舒张到90%的时程（选择90%而非100%是为了避免收缩时杂乱信号的干扰）。用fura-2/AM（0.5μM）将心肌细胞固定15分钟,然后用双激发荧光光电系统（Ionoptix）进行荧光标记。然后将心肌细胞放到奥林巴斯Ⅸ-70倒置显微镜,在40倍油镜下观察图像。细胞内Ca²⁺浓度的变化通过在360/380两个波长下fura-2荧光强度（fura-2 fluorescence intensity,FFI）比来推断;荧光延迟时间用细胞内Ca²⁺的清除率来衡量,如适合程序的单指数（single-exponential）曲线或双指数（bi-exponential）曲线。细胞内活性氧（ROS）的产生的测定是通过分析细胞内5-甲基-2’,7-二氢荧光素双乙酸钠的氧化所造成的荧光素强度的变化来推断的。用蛋白羰基含量实验来检测蛋白的损伤。心肌细胞的凋亡使用caspase-3活性荧光测定法。应激信号蛋白p38,JNK和ERK及与凋亡有关蛋白Bax,Bcl-2以及内质网应激的蛋白标志物GRP78和Gadd153用免疫印迹法测定。GAPDH做为上样控制。数据均用均数±标准误表示。多组之间使用GraphPad Prism 4中方差分析（ANOVA）与Tukey post hoc检验。P值小于0.05认为有统计学意义。结果ELISA方法测定血清中IGF-1的表达,结果示在心脏特异性IGF-1过表达小鼠和FVB小鼠中急性脂多糖处理不会影响其血循环IGF-1的水平。单心肌细胞功能测定指标显示在FVB-LPS组中细胞收缩峰值与收缩舒张最大速率较IGF-1-LPS组有明显减小,并且有舒张时限的延长,这一些改变与细胞内Ca²⁺衰减改变有关。活性氧簇,蛋白羰基及心肌细胞凋亡在LPS处理后的小鼠中均有增加。免疫印迹分析显示与FVB组比较在FVB-LPS组中p38,JNK和Bax以及内质网应激的蛋白标志物GRP78和Gadd153均有激活,但ERK和Bcl-2没有改变。有趣的是,这一些因脂多糖诱导的改变,如心肌细胞收缩功能与细胞内Ca²⁺处理功能不全,活性氧簇的产生,蛋白损伤,心肌细胞凋亡,应激信号及内质网应激的激活均被心脏特异过表达IGF-1所改善或减轻。体外的实验也证实了IGF-1（50 nM）可减轻因脂多糖（1μg/ml）诱导的单心肌细胞收缩功能不全。结论1.在心脏特异性IGF-1过表达小鼠和FVB小鼠中急性脂多糖处理不会影响其血循环IGF-1的水平;2.体外实验表明IGF-1至少部分对脂多糖诱导的心肌细胞收缩功能与细胞内Ca²⁺处理功能不全有改善;3.体内实验表明IGF-1本身不会对小鼠心脏收缩功能与细胞内Ca²⁺产生影响,心脏特异性过表达IGF-1可减轻由脂多糖诱导的小鼠心脏收缩功能与细胞内Ca²⁺处理功能不全;4.IGF-1减少由脂多糖诱导的活性氧簇的产生及蛋白损伤和心肌细胞凋亡;5.IGF-1减轻脂多糖诱导的应激信号蛋白,凋亡蛋白及内质网应激标志蛋白的激活。更多还原

【Abstract】 Background and ObjectiveWith the widespread application of percutaneous coronary intervention（PCI） and coronary artery bypass grafting（CABG） in ischemia heart disease,myocardial ischemia and reperfusion（I/R） injury received intensive attention from heart disease experts.In the past few years,except that applying medicines and the gene treatment method,growing evidence from animal experiments and even clinical observations indicates that Toll-like receptor 4 contributes significantly to subsequent functional and cellular injury through a variety of pathological pathways such as in liver or kidney ischemia and reperfusion,and it has been one of the most intensely studied topics in biomedical research and clinical practice.However,the signaling mechanisms in the affection of Toll-like receptor 4 in cardiac ischemia and reperfusion（I/R） injury are incompletely understood.Myocardial ischemia resulted from blockade in the coronary arteries caused by atherosclerosis can impair oxygen delivery.Toll-like receptor 4（TLR-4）,a proximal signaling receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens is expressed in the heart and vasculature.TLR-4 binds lipopolysaccharide（LPS） from the gram-negative bacteria and autogenous ligands such as heat shock proteins and fibronectin which are released during oxidative stress, following ischemic injury.Increased TLR-4 expression has been observed in human heart failure and ischemic hearts.Mitogen-activated protein kinases（MAPKs） such as p38,ERK,and JNK are located downstream of TLR-4.Ischemia activates p38,which demonstrated negative inotropic and restrictive diastolic effects.The AMP-activated protein kinase（AMPK） is an energy-sensing enzyme that can be activated by ischemia,and it has been found that AMPK plays an important role in cardioprotection against ischemic injury.In cardiac muscle,AMPK activity is increased by stimuli such as exercise,hypoxia,ischemia,and stress.It plays a central role in monitoring the cellular energy status and controlling energy production and consumption.Intriguingly,it has been reported that AMPK protects cardiomyocytes against hypoxic injury by attenuation of endoplasmic reticulum（ER） stress.In eukaryotic cells,the ER plays a vital role in the maturation,processing and transporting secretory and membrane associated proteins.The ER is exquisitely sensitive to alteration in homeostasis,with perturbations in the environment resulting in a condition known as ER stress.However,the molecular mechanisms behind TLR-4 deficiency-induced cardioprotection against ischemia/reperfusion damage remain unclear.Here we examined whether the cardioprotection of TLR-4 deficient hearts against ischemia/reperfusion injury mediated by AMPK signaling pathways and what’s the role of TLR-4 downstream,the MAPK signaling pathway.We also try to address the relationship between ischemic stress and ER stress in the heart.Materials and methodsIn vivo regional ischemia was induced by occlusion of the left anterior descending（LAD） coronary artery in wild type（WT） C3H/HeN and TLR-4 deficient C3H/HeJ mice that were purchased from the Jackson Laboratory（Bar Harbor,ME）. C3H/HeJ do not express functional TLR-4 because of naturally occurring mutations in the TLR-4 gene(Tlr4^Lps-d).C3H/HeN and C3H/HeJ Mice were anesthetized with ketamine（95 mg/kg） and xylazine（40 mg/kg） intraperitoneally,incubated,and ventilated with a respirator.After thoracotomy,a suture was placed to ligate the proximal left anterior descending（LAD） coronary artery for different time requirement.Control mice underwent sham thoracotomy.Mouse hearts were divided into 8 experimental groups:（1） C3H/HeN mice（WT） sham（n=7）;（2） C3H/HeN（WT） I/R 60 rains（n=7）;（3） C3H/HeN（WT） I/R 120 rains（n=7）;（4） C3H/HeN（WT） ischemia 15 mins（n=7）;（5） C3H/HeJ（TLR-4） sham（n=7）;（6） C3H/HeJ（TLR-4） I/R 60 mins（n=7）;（7） C3H/HeJ（TLR-4） I/R 120 mins（n=7） and（8） C3H/HeJ（TLR-4） ischemia 15 mins（n=7）.After ischemia/reperfusion,the myocardial infarct size was determined by means of a double-staining technique.Evan’s blue stained area,TTC stained area and TTC stained negative area were measured digitally using Image Pro Plus software.The degree of apoptotic activity was assessed by measuring caspase-3 activity in tissue homogenates using a commercially available fluorimetric assay.In addition,TUNEL assessment of myonuclei positive for DNA strand breaks was determined using a fluorescence detection kit.Heart tissue was analyzed for tumor necrosis factor-α（TNF-α）,interleukin（IL）-1βand IL-6 levels as determined by Enzyme-linked immunosorbent assay（ELISA）.Immunoblotting analysis was performed to assess the activation of AMPK and its downstream protein ACC and eEF2 and expression of the stress signaling the mitogen-activated protein kinase （MAPK） molecules JNK,p38 and ERK as well as the ER stress markers GRP78, Gadd153 and IRE1α.Data were Means±SEM.Differences between groups were assessed by variance （ANOVA） followed by Newman-Keuls post hoe test using GraphPad Prism 4.A P value less than 0.05 was considered statistically significant.ResultsThe extent of myocardial infarction was measured with dual staining to define the degree of necrosis within the ischemic region at risk.C3H/HeJ hearts demonstrated significantly smaller infarct size than C3H/HeN hearts.Ischemia and reperfusion can initiate pathways leading to cardiac apoptosis.Our data showed that there was an increase in caspase-3 activity cardiac myocytes in C3H/HeN hearts compared with C3H/HeJ hearts,suggesting greater activation of apoptotic pathways in C3H/HeN hearts.In addition,confocal microscopy revealed a greater number of TUNEL-positive nuclei following ischemia/reperfusion in C3H/HeN hearts compared with C3H/HeJ hearts.We determined proinflammatory cytokines TNF-α,IL-1βand IL-6 levels in serum.For all cytokines,significantly higher serum levels were seen in WT and C3H/HeJ hearts following MI/R compared to their shams.As would be expected,for WT hearts,all cytokines were higher with I/R vs.C3H/HeJ hearts. These results indicate that TLR-4 deficiency leads to a cardioprotective effect to prevent ischemia and reperfusion injury.To assess whether AMPK signaling pathway is involved in the resistance to I/R injury in C3H/HeJ mice,we first examined the AMPK phosphorylation of C3H/HeJ and C3H/HeN hearts during in vivo regional of I/R.The phosphorylation at Thr172 and activation of AMPK was significantly augmented in C3H/HeJ hearts as compared to that in C3H/HeN hearts.Moreover,the phosphorylation of the AMPK downstream targets,acetyl-CoA-carboxylase（ACC） and eukaryotic elongation factor 2（eEF2）, were also enhanced in C3H/HeJ hearts compared to C3H/HeN hearts.In order to determine the role of the TLR-4 downstream effectors,such as MAPKs in ischemic injury,we measured the activation of p38,JNK and ERK.The results demonstrated that ischemia time-dependently stimulated p38,JNK and ERK activation in both C3H/HeN and C3H/HeJ hearts as manifested by phosphorylation immunoblots.Intriguingly,p38 and JNK signaling were blunted in C3H/HeJ hearts compared to C3H/HeN hearts during ischemia.In contrast,ERK signaling was enhanced in C3H/HeJ hearts compared to C3H/HeN hearts during ischemia.It has been reported that ischemia leads to the accumulation of misfolded proteins in the endoplasmic reticulum（ER）,causing ER stress.Under conditions of ER stress,inhibition of protein synthesis and up-regulation of ER chaperone expression to reduce the misfolded proteins in the ER.We therefore examined whether TLR-4-deficiency affected ischemia and reperfusion induced ER stress in the hearts.The results demonstrated that ischemic treatment promoted ER stress,as shown by up-regulation of the ER chaperone,Gadd153/CHOP andGrp78/BiP,and the integral protein of ER membrane,IRE1α,while up-regulation in all these ER stress makers was blunted in C3H/HeJ hearts during I/R.Conclusions1.TLR-4 deficiency reduces infarct size after Ischemia/Reperfusion;2.Reduced Apoptosis in TLR-4-deficient Hearts during Ischemia/Reperfusion;3.TLR-4 deficiency reduces proinflammatory cytokines TNF-α,IL-1βand IL-6 levels in serum;4.Augmented AMPK Activation in TLR-4-deficient Mice during Ischemia/Reperfusion;5.Impaired Activation of MAPK Signaling in C3H/HeN vs.C3H/HeJ during Ischemia/Reperfusion;6.Resistance to Cardiac ER Stress in TLR-4-deficient Heart during Ischemia/Reperfusion. Background and ObjectiveInsulin-Like Growth Factor I（IGF-1） is essential for the maintenance of bodily structure and function.IGF-1 participates in the regulation of tissue remodeling, glucose metabolism,insulin sensitivity,lipid profile,myocardial growth and myocardial function in both physiological and pathophysiological conditions.The deficiency of which is associated with altered body composition,cytokine and neuroendocrine activation,cardiac atrophy and impaired cardiac function.A fall in the serum IGF-1 level,which often accompanies the biological aging process,leads to abnormal body composition and metabolism.The severely impairment of lifespan in IGF-1-deficient Ames dwarf mice compromised cardiac excitation-contraction coupling in cardiomyocytes.Similarly,patients with IGF-1 deficiency exhibit cardiac dysfunctions reminiscent of aging,which is mainly manifested as reduced left ventricular mass,ejection fraction and diastolic filling.Lipopolysaccharide（LPS） is a major component of the outer membrane of Gram-negative bacteria,contributing greatly to the structural integrity of the bacteria, and protecting the membrane from certain kinds of chemical attack.LPS also increases the negative charge of the cell membrane and helps stabilize the overall membrane structure.It is of crucial importance to gram negative bacterial cells;death results if it is mutated or removed.LPS is an endotoxin,and induces a strong response from normal animal immune systems.It acts as the prototypical endotoxin because it binds the CD14/TLR4/MD2 receptor complex,which promotes the secretion of pro-inflammatory cytokines in many cell types,but especially in macrophages.Severe sepsis remains a major cause of death in hospitalized patients in the Unites States,and these deaths are caused frequently by shock,which results in multiple organ failure. Myocardial depression is a common feature of endotoxemia in patients and in experimental model of lipopolysacharide（LPS）-induced sepsis,and has been recognized as a key component contributing to the development of septic shock.Low levels of LPS may mediate vascular inflammation in atherosclerosis.Low circulating levels of insulin-like growth factorⅠ（IGF-1） are found in sepsis although the influence of IGF-1 on septic cardiac defect is still unknown.This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca²⁺ dysfunction,activation of stress signal and endoplasmic reticulum（ER） stress.Materials and methodsWeight-（20-25g） and age-（4-5 months of age） matched adult male FVB and IGF-1 transgenic mice were used in this study.All animals were kept in our institutional animal facility with free access to standard laboratory chow and tap water. On the day of experimentation,both FVB and IGF-1 transgenic mice were injected intraperitoneally with 4 mg/kg Escherichia Coli LPS dissolved in sterile saline or an equivalent volume of pathogen-free saline（for control groups）.The dosage of LPS injection was chosen based on previous observation of overt myocardial dysfunction without significant mortality.Six hrs following LPS challenge,mice were sacrificed for experimentation.Serum IGF-1 was analyzed using an enzyme-linked immunosorbent assay（ELISA）in accordance with manufacturer’s recommendations （R & D System Inc,Minneapolis,MN,USA）.Cardiomyocytes were isolated by Liberase Blendzyme 4 for 20 mins using Langendorff system.Myocyte yield was～70%which was not overtly affected by either LPS or IGF-1.Only rod-shaped myocytes with clear edges were selected for mechanical and intracellular Ca²⁺ transient studies.To elucidate the cardiac-specificity, if any,of LPS-induced cardiomyocyte mechanical response,a sub-lethal dose of Escherichia coli LPS（1μg/ml） was administered to cardiomyocytes isolated from FVB mice.The cells were incubated with LPS for 4-6 hrs in the presence or absence of exogenous recombinant IGF-1（50 nM）.The mechanical properties of cardiomyocytes were assessed using a SoftEdge MyoCam system（IonOptix Corporation,Milton,MA,USA）.An IonOptix SoftEdge software was used to capture changes in cell length during shortening and relengthening.Cell shortening and relengthening were assessed using the following indices:peak shortening（PS）-indicative of the amplitude a cell can shorten during contraction;maximal velocities of cell shortening and relengthening（±dL/dt）-indicative of peak ventricular contractility;time-to-PS（TPS）-indicative of systolic duration;time-to-90%relengthening(TR₉₀)-indicative of diastolic duration（90% rather 100%relengthening was used to avoid noisy signal at baseline concentration）.Myocytes were loaded with fura-2/AM（0.5μM） for 10 mins and fluorescence measurements were recorded with a dual-excitation fluorescence photo multiplier system（Ionoptix）.The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of fura-2 fluorescence intensity（FFI） at two wavelengths（360/380）. Fluorescence decay time was measured as an indication of the intracellular Ca²⁺ clearing rate.Both single and bi-exponential curve fit programs were applied to calculate the intracellular Ca²⁺ decay constant.Production of cellular ROS was evaluated by analyzing changes in fluorescence intensity resulting from oxidation of the intracellular fluoroprobe 5-（and -6）-chloromethyl-2’,7’-dichlorodihydrofluorescein diacetate（CM-H₂DCF-DA, Molecular Probes,Eugene,OR,USA）.The carbonyl content of protein was determined using protein carbonyl assay.The degree of apoptotic activity was assessed by measuring caspase-3 activity in tissue homogenates using a commercially available fluorimetric assay.Expression of the stress signaling molecules JNK,p38 and ERK,the apoptotic proteins Bax and Bcl-2 as well as the ER stress markers GRP78 and Gadd153 was assessed using Western blotting.GAPDH was used as the internal loading control.Data were Means±SEM.Differences between groups was assessed by variance （ANOVA） followed by Tukey post hoc test using GraphPad Prism 4 or student’s t-test wherever appropriate.Ap value less than 0.05 was considered statistically significant.ResultsAcute LPS treatment failed to affect plasma IGF-1 levels in either IGF-1 or FVB mice.Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening as well as prolonged duration of relengthening in LPS-treated FVB cardiomyocytes associated with reduced intracellular Ca²⁺ decay. Accumulation of ROS,protein carbonyl and apoptosis were elevated following LPS treatment.Western blot analysis revealed activated p38 and JNK,upregulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2.Total protein expression of p38,JNK and ERK was unaffected by either LPS or IGF-1.Interestingly,these LPS-induced changes in mechanical and intracellular Ca²⁺ properties,ROS,protein carbonyl,apoptosis,stress signal activation and ER stress markers were effectively ablated by IGF-1.In vitro LPS exposure（1μg/ml） produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting,which was alleviated by exogenous IGF-1（50 nM）.Conclusions:1.Acute LPS treatment failed to affect plasma IGF-1 levels in either IGF-1 or FVB mice;2.In vitro study shows that LPS-elicited cardiomyocyte defect and IGF-1-offered cardioprotection may be attributed,at least in part,to changes in intrinsic cardiomyocyte properties; 3.IGF-1 itself did not elicit any overt effect on mechanical and intracellular Ca²⁺ properties.Overexpression of the growth factor significantly attenuated LPS-induced cardiomyocyte mechanical and intracellular Ca²⁺ dysfunction;4.IGF-1 reduced LPS-elicited effects on ROS generation,protein damage and apoptosis:5.IGF-1 ablated LPS-induced Stress signaling activation,apoptotic proteins and ER stress.These data collectively suggested a beneficial of IGF-1 in the management of cardiac dysfunction under sepsis.更多还原