线粒体Cx43预防兔心肌缺血/再灌注损伤作用机制的研究
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摘要
缺血心肌再灌注过程中常易发生缺血再灌注损伤(ischemia reperfusion injury, IRI),心肌IRI是临床上常见的一种心肌损害现象,并且其机制复杂。目前心肌IRI保护的干预措施主要包括缺血预处理(ischemic preconditioning,IP)、药物预处理和缺血后处理(ischemic postconditioning,PC),PC、IP和药物预处理等存在一些共同的保护机制。研究发现PC具有明显的心肌保护作用,但是其确切机理至今仍未完全阐明。近年来在体、离体的研究证实IRI早期引起Cx43脱磷酸化,缝隙连接(Gap junction,GJ)开放,引起坏死物质的传播导致梗死加重。GJ参与IRI,以GJ为靶点的抗IRI药物可能是一个新的发展方向,Heptanol(庚醇)作为GJ的解耦联剂,在临床上有潜在运用价值。2005年Schulz等首次在心肌线粒体上发现缝隙连接蛋白43(connexin 43,Cx43)的表达,线粒体Cx43在IP中作用也成热点研究之一。新近研究提示心肌线粒体以及线粒体Cx43在心肌IRI损伤以及IP心肌保护中具有非常重要的作用。同时在IP心肌保护作用中线粒体ATP敏感钾通道(mitoKATP)参与GJ调控。
而Cx43在PC与Heptanol预处理中少有研究,尤其是线粒体中Cx43在PC和Heptanol预处理中作用至今未见报道。故我们设想线粒体Cx43在PC和Heptanol预处理保护缺血再灌注心脏中也同样起重要作用, PC与Heptanol保护的机制可能也是通过激活mitoKATP,使细胞膜和线粒体Cx43的数量、分布及功能发生变化,减少损伤因子在细胞间的扩散,同时增强细胞线粒体自身的稳定及抗损伤能力,从而达到抗IRI损伤的效益。本实验旨在探讨线粒体及线粒体Cx43是否参与PC和Heptanol的心肌保护作用及其可能机制。
本研究建立兔心肌IRI模型,给予PC和Heptanol预处理,并应用5-羟葵酸(5-HD)即线粒体ATP敏感性钾通道(mitoKATP)特异性阻滞剂,观察PC和Heptanol对IRI兔心肌保护作用及线粒体Cx43在其中所起的作用。本课题共分为四个部分加以研究,即(1)PC及Heptanol预处理对兔IRI的影响;(2)PC及Heptanol预处理对兔IRI心肌细胞凋亡与线粒体结构及功能的影响;(3)PC及Heptanol预处理对兔IRI的细胞膜及线粒体Cx43影响;(4)mitoKATP和线粒体Cx43在缺血后处理和Heptanol预处理相关IRI中的作用。
第一部分缺血后处理及Heptanol预处理对兔心肌缺血/再灌注保护作用
目的:建立兔IRI模型,观察PC、Heptanol预处理对兔IRI的保护作用。
方法:80只新西兰大白兔随机分为5组,雌雄不分,分别为假手术组(Sham)、心肌缺血/再灌注模型组(IR)、缺血预处理组(IP),缺血后处理组(PC)以及Heptanol预处理组(HT),每组16只。假手术组左冠状动脉前降支近端穿线但不结扎,观察4h,其余4组则结扎30min后,松开结扎线,予4h再灌注。分别于结扎前、结扎后30min、再灌注30min、再灌注2h、再灌注4h取颈动脉血测定血浆磷酸肌酸激酶同工酶(CK-MB),肌钙蛋白(cTnI)以及超氧化物歧化酶(SOD)与丙二醛(MDA)含量以及通过颈动脉插管测定上述5个时点的血流动力学指标。再灌注结束后,Even’s和TTC染色法测心梗面积,HE染色观察各组心肌组织结构变化,电镜观察各组心肌细胞超微结构的变化。取心肌组织,制备匀浆液,测定SOD与MDA含量。
结果:1.心肌酶学:与Sham组比较,其余各组CK-MB、cTnI值在结扎30min、再灌30min、再灌2h及再灌4h后明显升高(P <0.01)。与IR组比较,IP组、PC组和HT组的CK-MB、cTnI值在再灌30min、再灌2h和再灌4h后均明显下降(P <0.01)。2.血流动力学指标:各组术前均无差异。术后,与Sham组比较,各处理组结扎后30min、再灌注30min、再灌注2h、再灌注4h的HR、MAP、±dp/dtmax均明显降低,LVEDP明显升高,均有显著性差异(P <0.01);与IR组比较,IP、PC和HT组的MAP、±dp/dtmax(P <0.05)和LVEDP(P <0.01)各指标在再灌注2h、4h明显改善,有统计学意义。3.心梗面积: IP,PC和HT组的心梗面积明显小于IR组,差异有统计学意义(P <0.01)。3.心肌组织结构的变化和心肌超微结构的变化:光镜与电镜显示结果,与Sham组比较,IR组心肌细胞损伤明显;与IR组比较,IP、PC和HT组心肌细胞损伤减轻明显。4.心肌组织匀浆及血清中SOD与MDA变化:缺血再灌注4h后,与Sham组比较,IR组SOD活性明显降低, MDA明显升高,差异有统计学意义(P <0.01)。与IR组比较,IP和PC组SOD活性明显升高,MDA明显减低,差异有统计学意义(P <0.01);而HT组SOD与MDA活性改变无显著性差异(P >0.05)。
结论:PC与Heptanol预处理对兔心肌IRI的具有保护作用。
第二部分缺血后处理、Heptanol预处理对兔心肌缺血/再灌注心肌细胞凋亡及线粒体结构、功能的影响
目的:建立兔缺血再灌注模型,观察PC、Heptanol预处理对兔心肌IRI心肌凋亡、线粒体结构即功能的影响。
方法:80只新西兰大白兔随机分为5组,动物模型制备、分组及纳入例数均与第一部分相同。采用TUNEL法检测各组缺血心肌细胞凋亡,用透射电镜观察心肌线粒体的超微结构改变,同时检测线粒体膜电位、Ca2+浓度、丙二醛(MDA)浓度、超氧化物岐化酶(SOD)活性的改变。
结果:与IR组比较,PC、HT和IP组兔心肌细胞凋亡减少(P <0.01),心肌线粒体形态结构改变明显减轻(P <0.01);线粒体跨膜电位明显升高(P <0.01)、线粒体Ca2+浓度(P <0.05)明显下降,差异有统计学意义;PC、HT和IP组比较差异无统计学意义(P >0.05)。与IR组比较,HT组SOD与MDA改变差异无统计学意义(P >0.05);IP组和PC组SOD活性明显升高、MDA浓度均下降,差异有统计学意义(P <0.05)。
结论:PC、Heptanol可能通过提高线粒体跨膜电位、减轻线粒体钙超载以致改善缺血再灌注心肌细胞损伤、减轻心肌凋亡;PC可降低线粒体氧自由基水平,而与PC心肌保护机制不同的是,HT不能降低线粒体氧自由基水平。
第三部分缺血后处理、Heptanol预处理对兔心肌缺血/再灌注损伤中细胞膜及线粒体Cx43的影响
目的:探讨PC、Heptanol对兔IRI中细胞膜及线粒体Cx43的影响。
方法:兔80只,建立心肌IRI模型,随机分5组,每组16只:假手术组(Sham组)、缺血再灌注组(IR组)、缺血预处理组(IP组)、缺血后处理组(PC组)和Heptanol预处理(HT组)。应用电镜观察各组闰盘结构变化;应用免疫组化、免疫荧光法检测细胞膜Cx43-GJ分布及含量变化;采用差速离心法提取各组缺血区心肌线粒体蛋白,应用Western blot检测线粒体Cx43蛋白含量。进一步从转录水平采用RT-PCR检测Cx43mRNA表达。
结果:(1)闰盘结构变化与Sham组比较,IR组闰盘及GJ结构不清,可见移位、断裂。与IR组比较,IP、PC和HT组的闰盘病变明显减轻。(2)细胞膜Cx43、GJ与Sham组比较,IR组GJ重构明显,胞膜Cx43含量明显减少(P <0.01)。与IR组比较,IP、PC和HT组的GJ重构明显减轻,并且胞膜Cx43含量增加(P <0.01)。(3)线粒体Cx43蛋白与Sham组比较,IR组线粒体Cx43蛋白显著下降(P <0.01);与IR组比较,PC组、HT组和IP组心肌线粒体Cx43明显升高(P <0.01)。
结论:PC、Heptanol预处理可以减轻IRI引起的闰盘损伤、细胞膜和线粒体Cx43的减少及改善GJ重构,细胞膜与线粒体Cx43可能参与了PC和Heptanol预处理的心肌保护作用。
第四部分线粒体ATP敏感钾通道和线粒体Cx43在缺血后处理和Heptanol预处理相关缺血/再灌注损伤中的作用
目的:探讨线粒体ATP敏感钾通道和线粒体Cx43在PC和Heptanol保护兔IRI中的作用。
方法:兔112只,建立IRI模型,随机分7组,每组16只:假手术组(Sham组)、缺血再灌注组(IR组)、缺血后处理组(PC组)和Heptanol预处理(HT组)、缺血后处理组+5HD组(PC+5HD组)和Heptanol预处理+5HD组(HT+5HD组)。假手术组左冠状动脉前降支近端穿线但不结扎,观察4h,其余4组则结扎30min后,予4h再灌注。取血测定结扎前、结扎后30min、再灌注30min、再灌注2h、再灌注4h血浆CK-MB、cTnI以及SOD与MDA含量。实验结束后,Even’s和TTC染色法测心梗面积。电镜观察心肌细胞超微结构尤其是线粒体与闰盘结构的变化。TUNEL法检测缺血心肌细胞凋亡。免疫荧光检测胞膜cx43-GJ变化以及胞膜Cx43含量,差速离心法提取线粒体,并检测线粒体膜电位、Ca2+浓度、MDA浓度、SOD活性的改变。应用Western Blot检测线粒体Cx43蛋白含量。进一步用RT-PCR检测心肌组织中Cx43mRNA改变。
结果:再灌注4h末,IP、PC和HT组的血浆CK-MB、cTnI及心梗面积明显低于IR、PC+5HD和HT+5HD组(P <0.01)。与IR、PC+5HD和HT+5HD组比较,IP、PC和HT组兔心肌细胞凋亡减少,心肌、闰盘及线粒体形态结构改变明显减轻,线粒体跨膜电位、SOD活性明显升高,线粒体Ca2+浓度、MDA浓度均下降。免疫荧光检测细胞膜Cx43、GJ及Western Blot检测线粒体Cx43蛋白发现,与Sham组比较,IR、PC+5HD和HT+5HD组细胞膜与线粒体Cx43蛋白显著下降(P <0.01);与IR组比较,PC、HT、IP、PC+5HD和HT+5HD组心肌胞膜和线粒体Cx43明显升高(P <0.01和P <0.05))。与PC+5HD和HT+5HD组比较,IP、PC和HT组胞膜Cx43及线粒体Cx43蛋白明显升高(P <0.01)。
结论:PC、Heptanol预处理可以减轻IRI引起的闰盘损伤、改善GJ重构、减轻IRI所致的细胞膜及线粒体Cx43的含量,其可能通过提高线粒体跨膜电位、减轻线粒体钙超载、改善缺血再灌注心肌细胞凋亡等来达到对兔IRI的保护作用。细胞膜、线粒体Cx43可能参与了PC和Heptanol预处理的心肌保护作用,其机制与mitoKATP有关。PC也可降低线粒体氧自由基水平。
Myocardial ischemia reperfusion frequently prone to ischemia-reperfusion injury (IRI), myocardial ischemia-reperfusion injury is a common clinical phenomenon of myocardial injury and its mechanism complex.Myocardial protection interventions currently include ischemic preconditioning (IP), pharmacological preconditioning and ischemic postconditioning (PC) .In view of PC and IP, as well as pharmacological preconditioning, there are some similar protection mechanisms among the treatments mentioned above . Studies find that PC plays a significant role in myocardial protection, but its exact mechanism has not yet to be clarified. The vivo and vitro studies confirmed that dephosphorylation of Cx43, and the opening of gap junction(GJ) channel at the early stage of IRI, which cause the widespread of necrosis agents and aggravate the process of infarction.GJ participate in the process of IRI, take GJ as the target of anti-IRI therapy may be a new direction of development.In 2005, Schulz found the expression of connexin 43(Cx43) in myocardial mitochondrial for the first time, and recently, the role of mitochondrial Cx43 in IP is also one of the hot-top studies. Recent researches find that heart mitochondria and mitochondrial Cx43 play a very important role in myocardial IRI and IP protection. While IP myocardial protection of mitochondrial ATP-sensitive potassium channel (mitoKATP) involved in regulation of GJ.
And Cx43 in PC and heptanol pretreatment has the few studies, particularly the mitochondria Cx43 role in the PC and heptanol preconditioning has not been reported. We assume that mitochondrial Cx43 in PC and heptanol preconditioning also plays an important role. PC and heptanol protection mechanisms may also by activating mitochondrial-sensitive potassium channel,so that there are certain changes in quantities,distribution and function of cell membrane and mitochondrial Cx43,which can prevent the disffusion of injury agents,enhance the stabilization of mitochondrial and anti-injure competence,then attenuate the hazardous effect of IRI. This study are designed to investigate whether the mitochondria and mitochondrial Cx43 are involved in PC and heptanol on myocardial protection and its possible mechanism.
In this study, we built rabbit myocardial IRI model, with PC and Heptanol pretreatment and 5 - hydroxy caproic acid (mitochondrial ATP-sensitive potassium channel specific blocker,5HD) treatment.The protective effect of PC and Heptanol on IRI and their impact on mitochondrial Cx43 will be observed,then explore the possible protective mechanisms of mitochondrial Cx43 in the PC and Heptanol treatment on rabbits with acute myocardial IRI .The topic to be studied is divided into four parts, namely (1)Impact of PC and Heptanol pretreatment on rabbit myocardial IRI; (2) Impact of PC and Heptanol pretreatment on myocardial apoptosis and mitochondrial structure and function in rabbit myocardial IRI; (3) Impact of PC and Heptanol pretreatment on Cx43 of membrane and mitochondria in rabbit myocardial IRI; (4) Impact of PC and Heptanol pretreatment involves mitochondrial ATP-sensitive potassium channel,and the relationship between the expression of mitochondria Cx43 suffered from ischemia-reperfusion .
Part 1 Effection of Ischemic postconditioning and heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits
Objective: To investigate the protection of ischemic postconditioning and Heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits.
Methods:80 New Zealand white rabbits were randomly divided into five groups, namely sham-operated group (Group Sham), myocardial ischemia / reperfusion model group (Group IR), ischemic preconditioning group (Group IP), ischemia and postconditioning group (PC) and heptanol pretreatment group (Group HT), n = 16. Sham-operated group, thread the proximal left anterior descending(LAD) coronary artery without ligation and observed 4h, while in other groups, ligated LAD for 30min, then released for reperfusion for 4h. Determin the concentration of plasma creatine kinase isoenzyme (CK-MB), troponin (cTnI), SOD and MDA level prior to ligation ,ligation taken 30min, reperfusion 30min, reperfusion 2h and reperfusion 4h. After reperfusion, myocardial infarction areas were measured by Evans blue and TTC staining.SOD and MDA level were measured by prepared homogenate of Myocardial tissue. The SOD and MDA in serum and myocardium were measured respectively.HE stained first,then observed ultrastructural changes of myocodrial by electron microscope.
Results: 1. Enzymatic markers: compared to sham group, the values of CK-MB, cTnI in other groups at the points of ligation 30min, reperfusion 30min, reperfusion after 2h and 4h reperfusion were significantly highe (P <0.01). Compared with the Group IR, the Group IP, PC and HT CK-MB, cTnI values significantly decreased at reperfusion 30min, reperfusion after 2h and 4h reperfusion (P <0.01). 2. Hemodynamics: there was no statistical significance in HR, MAP, LVEDP + dp / dtmax and -dp/dtmax before ischemia among different groups. After reperfusion, compared to Group sham, HR, MAP, + dp/dtmax, -dp/dtmax in treatment groups after ligation 30min, reperfusion 30min, reperfusion 2h, reperfusion 4h decreased, but LVEDP increased significantly (P <0.01);compared to Group IR, MAP, + dp / dtmax, -dp/dtmax (P <0.05) and LVEDP (P <0.01) indexes at the points of reperfusion 2h, 4h significantly improved in IP, PC and HT groups. 3. Infarct area: Group IP (18.97%±2.8%), Group PC (19.07%±4.9%) and the Group HT (20.01%±3.9%) of myocardial infarct areas are significantly smaller than Group IR (35.67%±5.8%,P <0.01). 4. Myocardial structure modification and changes of myocardial ultrastructure: light microscope and electron microscope results, compared to Group sham, there is a significant myocardial injury in Group IR; in contrast of Group IR, IP, PC and HT receive a slighter myocardial cell injury. 5. Myocardium tissue and changes in serum SOD and MDA: 4h after ischemia, compared to Group sham,Group IR SOD activity significantly decreased, while MDA significantly increased(P <0.01). Compared to Group IR, Group IP and Group PC have significantly higher SOD activitives, MDA significantly decreased(P <0.01); there is no significant differences of SOD and MDA in HT group (P >0.05).
Conclusion: Ischemic postconditioning and Heptanol pretconditioning can protect the heart from IRI injury. But Heptanol preconditioning has no effect on SOD activity and MDA content. This is the difference from the Ischemic postconditioning about pretecting IRI injury.
Part 2 Effect of ischemic postconditioning and Heptanol on apoptosis and structural changes of mitochondria in ischemia reperfusion injury of rabbit
Objective:To investigate the effects of ischemic postconditioning and Heptanol preconditioning on apoptosis and structural and functional changes of mitochondria induced by IRI of rabbits in vivo, in order to discuss the mechanisms of ischemic postconditioning and Heptanol preconditioning from the view of mitochnodial.
Methods: Eighty rabbits were divided randomly into five groups:sham operation group(Group Sham),ischemic reperfusion group (Group IR),ischemic preconditioning group (Group IP), ischemic postconditioning group (Group PC) and Heptanol preconditioning group (Group HT) ,with sixteen rabbits in each group.All rabbits were sacrificed after reperfusion for 4h.The heart was quickly removed for observing the structure of mitochondria and measurement of the apoptosis fraction by TUNEL. We observed ultrastructural changes of myocardium under electron microscope and examined mitochondrial membrane potential and Ca2+ concentration, MDA content and SOD activity of myocardial mitochondria.
Results: (1)The results of cardiac muscle cell apoptosis index:TUNEL positive cells obviously increased in Group IR, and with an intensive distribution. The AI of five groups were respectively Group sham(0.12%±0.07% ), Group IR(33.2%±1.2%),Group IP(14.4%±2.3%), Group PC(15.6%±1.9%)and Group HT(15.8%±1.8%) .The TUNEL positive cells of Group IP,PC and HT are obviously less than Group IR and the difference was significant(P <0.01). (2) The changes of mitoehondrial ultrastrueture: Compared to Group IR, in Group IP , Group PC and Group HT, the damages of mitoehondrial ultrastrueture were milder(P <0.01).(3) The results of mitochondrial function in five groups: Compared to Group IR, in Group IP, Group PC and Group HT, mitochondrial membrane potential(P <0.01) was significantly higher and Ca2+ concentration were much lower(P <0.05).No significant difference was found in indicators between Group IP , Group PC and Group HT. Compared to Group IR, MDA content and SOD activity of myocardial mitochondria in Group HT, no significant difference was found;But in Group IP and Group PC, MDA content were much lower and SOD activity was significantly higher((P <0.05).
Conclusion: Ischemic postconditioning and Heptanol preconditioning can protect the heart from IRI evidenced by rehabilitating mitochondrial ultrastructure,preventing apoptosis, increaseing mitochondrial membrane potential and alleviating Ca2+ overload in myocardial mitochondria. Be different from HT, PC can reduce the levels of oxygen free radicals in mitochondrial. This is the difference between PC and HT in myocardial protection.
Part 3 Impact of Ischemic postconditioning and heptanol preconditioning on Cx43 of membrane and mitochondria in rabbit myocardial ischemia-reperfusion injury
Objective: To investigate the effects of ischemic postconditioning and Heptanol preconditioning on Cx43 of membrane and mitochondria in rabbit myocardial ischemia-reperfusion injury
Methods: 80 New Zealand white rabbits were randomly divided into five groups, namely sham-operated group (Group Sham), myocardial ischemia / reperfusion model group (Group IR), ischemic preconditioning group (Group IP), ischemia postconditioning group (Group PC) and heptanol pretreatment group (Group HT), n = 16. All rabbits were sacrificed after reperfusion for 4h. Application of electron microscopy to illustrate the modification of intercalated disk; application of immunohistochemistry and immunofluorescence staining to observe the changes of membrane distribution and the content of Cx43; mitochondrial protein was extracted using gradient centrifugation. The mitochondria Cx43 were detected by Western Blotting.Further detection Cx43mRNA expression from the transcription level by Reverse Transcription Polymerase Chain Reaction.
Results:Compared to Group IR, there were moderate disorganized GJ distribution in Group IP , Group PC and Group HT. Compared to Group Sham, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly decreased in Group IR (P <0.01); These results were further confirmed by Reverse Transcription–Polymerase Chain Reaction.No significant difference was found among Group IP ,Group PC and Group HT(P >0.05).
Conclusion: Ischemic postconditioning and Heptanol preconditioning can protect the heart from ischemia reperfusion injury, it can rehabilitate the injured intercalated disc, remodel its reconstruction and prevent Cx43 decreases induced by IRI both in membrane and mitochondria. Myocardial protection of PC and heptanol preconditioning may involve Membrane and mitochondrial Cx43.
Part 4
Impact of PC and heptanol pretreatment involved in mitochondrial ATP-sensitive potassium channel on Cx43 of mitochondria in rabbit ischemia-reperfusion injury
Objective: To investigate Impact of PC and heptanol pretreatment involved in mitochondrial ATP-sensitive potassium channel on mitochondria Cx43 in rabbit ischemia-reperfusion injury.
Methods: 112 Rabbits, established myocardial ischemia-reperfusion model, were randomly divided into seven groups: sham operation group (Group Sham), ischemia reperfusion group (Group IR), ischemic postconditioning group (Group PC), and Heptanol preconditioning +5HD group (Group PC +5HD) and heptanol pretreatment +5HD group (Group HT +5HD), n=16. Sham-operated group, thread the proximal left anterior descending coronary artery without ligation and observed for 4h, while the other six groups, after ligation for 30min, released the ligature, reperfused for 4h. Determined the concentration of carotid artery of plasma creatine kinase isoenzyme (CK-MB), troponin (cTnI), as well as SOD and MDA prior to ligation ,ligation for 30min, reperfusion for 30min, reperfusion for 2h and reperfusion for 4h. After reperfusion, myocardial infarction areas were measured by Evans blue and TTC staining, myocardial structure and ultrastructure were observed by HE staining and electron microscopy and especially focus on mitochondria and intercalated disc structure.TUNEL assayed apoptosis in ischemic myocardium. Immunofluorescence detected membrane changes in GJ and Cx43 membrane content. Extracted mitochondria by gradient centrifugation and detected mitochondrial membrane potential, Ca2+ concentration, malondialdehyde(MDA) concentration, superoxide dismutase (SOD) activity of myocardial mitochondria.Analyze the content of mitochondrial Cx43 protein by Western blotting and further detection Cx43mRNA expression from the transcription level by Reverse Transcription Polymerase Chain Reaction.
Results: (1) Plasma CK-MB and cTnI activity and Myocardial infarct areas: After IR 4h, compared to Group IR,Group PC+5HD and Group HT+5HD,the CK-MB,cTnI and myocardial infarct areas of IP, PC and HT were obviously smaller(P <0.01). (2) The results of myocardium cell apoptosis index: compared to Group IR, Group PC+5HD and Group HT+5HD,TUNEL positive cells obviously decreased in IP, PC and HT myocardium tissue (P <0.01). (3) The modification of myocardial ultrastructure, GJ and mitochondrial: compared to Group IR, Group PC+5HD and Group HT+5HD,the modification of myocardial ultrastructure, GJ and mitoehondrial of IP, PC and HT were milder(P <0.01). (4) The results of mitochondrial function in five groups: Compared to Group IR, Group PC+5HD and Group HT+5HD, in IP,PC and HT, mitochondrial membrane potential was significantly higher and Ca2+ concentration were much lower(P<0.01).No significant difference was found among IP , PC and HT groups. Compared to Group IR, there was no significant difference in MDA content and SOD activity of myocardial mitochondria in Group HT;But in Group IP and Group PC, MDA content were much lower and SOD activity was significantly higher(P <0.05).(5) Compared to Group IR, Group PC+5HD and Group HT+5HD,the disorganized gap junction distribution in IP , PC and HT groups were milder (P <0.01). Compared to Group Sham, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly decreased in IR, PC+5HD and HT+5HD groups(P <0.01); Compared to Group IR, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly increased in IP,PC and HT groups (P <0.01) and PC+5HD and HT+5HD groups (P <0.05) . Compared to PC+5HD and HT+5HD groups, the sarcolemma Cx43-GJ and the mitochondria Cx43 expression obviously increased in IP,PC and HT groups (P <0.01).No significant difference was found among IP , PC and HT groups (P >0.05).
Conclusion: PC, Heptanol pretreatment can reduce the intercalated discs injury caused by IRI, improving GJ reconstruction, to reduce IRI induced degradation of Cx43 cell membrane and mitochondria, which may be by increasing mitochondrial membrane potential, reducing mitochondrial calcium overload, improving myocardial apoptosis and protection of myocardial mitochondrial structure of rabbit IRI. Mitochondrial Cx43 may be involved in the PC and Heptanol preconditioning on myocardial protection, its mechanism related to mitochondrial ATP-sensitive potassium channel. PC can also reduce the level of oxygen free radicals.
而Cx43在PC与Heptanol预处理中少有研究,尤其是线粒体中Cx43在PC和Heptanol预处理中作用至今未见报道。故我们设想线粒体Cx43在PC和Heptanol预处理保护缺血再灌注心脏中也同样起重要作用, PC与Heptanol保护的机制可能也是通过激活mitoKATP,使细胞膜和线粒体Cx43的数量、分布及功能发生变化,减少损伤因子在细胞间的扩散,同时增强细胞线粒体自身的稳定及抗损伤能力,从而达到抗IRI损伤的效益。本实验旨在探讨线粒体及线粒体Cx43是否参与PC和Heptanol的心肌保护作用及其可能机制。
本研究建立兔心肌IRI模型,给予PC和Heptanol预处理,并应用5-羟葵酸(5-HD)即线粒体ATP敏感性钾通道(mitoKATP)特异性阻滞剂,观察PC和Heptanol对IRI兔心肌保护作用及线粒体Cx43在其中所起的作用。本课题共分为四个部分加以研究,即(1)PC及Heptanol预处理对兔IRI的影响;(2)PC及Heptanol预处理对兔IRI心肌细胞凋亡与线粒体结构及功能的影响;(3)PC及Heptanol预处理对兔IRI的细胞膜及线粒体Cx43影响;(4)mitoKATP和线粒体Cx43在缺血后处理和Heptanol预处理相关IRI中的作用。
第一部分缺血后处理及Heptanol预处理对兔心肌缺血/再灌注保护作用
目的:建立兔IRI模型,观察PC、Heptanol预处理对兔IRI的保护作用。
方法:80只新西兰大白兔随机分为5组,雌雄不分,分别为假手术组(Sham)、心肌缺血/再灌注模型组(IR)、缺血预处理组(IP),缺血后处理组(PC)以及Heptanol预处理组(HT),每组16只。假手术组左冠状动脉前降支近端穿线但不结扎,观察4h,其余4组则结扎30min后,松开结扎线,予4h再灌注。分别于结扎前、结扎后30min、再灌注30min、再灌注2h、再灌注4h取颈动脉血测定血浆磷酸肌酸激酶同工酶(CK-MB),肌钙蛋白(cTnI)以及超氧化物歧化酶(SOD)与丙二醛(MDA)含量以及通过颈动脉插管测定上述5个时点的血流动力学指标。再灌注结束后,Even’s和TTC染色法测心梗面积,HE染色观察各组心肌组织结构变化,电镜观察各组心肌细胞超微结构的变化。取心肌组织,制备匀浆液,测定SOD与MDA含量。
结果:1.心肌酶学:与Sham组比较,其余各组CK-MB、cTnI值在结扎30min、再灌30min、再灌2h及再灌4h后明显升高(P <0.01)。与IR组比较,IP组、PC组和HT组的CK-MB、cTnI值在再灌30min、再灌2h和再灌4h后均明显下降(P <0.01)。2.血流动力学指标:各组术前均无差异。术后,与Sham组比较,各处理组结扎后30min、再灌注30min、再灌注2h、再灌注4h的HR、MAP、±dp/dtmax均明显降低,LVEDP明显升高,均有显著性差异(P <0.01);与IR组比较,IP、PC和HT组的MAP、±dp/dtmax(P <0.05)和LVEDP(P <0.01)各指标在再灌注2h、4h明显改善,有统计学意义。3.心梗面积: IP,PC和HT组的心梗面积明显小于IR组,差异有统计学意义(P <0.01)。3.心肌组织结构的变化和心肌超微结构的变化:光镜与电镜显示结果,与Sham组比较,IR组心肌细胞损伤明显;与IR组比较,IP、PC和HT组心肌细胞损伤减轻明显。4.心肌组织匀浆及血清中SOD与MDA变化:缺血再灌注4h后,与Sham组比较,IR组SOD活性明显降低, MDA明显升高,差异有统计学意义(P <0.01)。与IR组比较,IP和PC组SOD活性明显升高,MDA明显减低,差异有统计学意义(P <0.01);而HT组SOD与MDA活性改变无显著性差异(P >0.05)。
结论:PC与Heptanol预处理对兔心肌IRI的具有保护作用。
第二部分缺血后处理、Heptanol预处理对兔心肌缺血/再灌注心肌细胞凋亡及线粒体结构、功能的影响
目的:建立兔缺血再灌注模型,观察PC、Heptanol预处理对兔心肌IRI心肌凋亡、线粒体结构即功能的影响。
方法:80只新西兰大白兔随机分为5组,动物模型制备、分组及纳入例数均与第一部分相同。采用TUNEL法检测各组缺血心肌细胞凋亡,用透射电镜观察心肌线粒体的超微结构改变,同时检测线粒体膜电位、Ca2+浓度、丙二醛(MDA)浓度、超氧化物岐化酶(SOD)活性的改变。
结果:与IR组比较,PC、HT和IP组兔心肌细胞凋亡减少(P <0.01),心肌线粒体形态结构改变明显减轻(P <0.01);线粒体跨膜电位明显升高(P <0.01)、线粒体Ca2+浓度(P <0.05)明显下降,差异有统计学意义;PC、HT和IP组比较差异无统计学意义(P >0.05)。与IR组比较,HT组SOD与MDA改变差异无统计学意义(P >0.05);IP组和PC组SOD活性明显升高、MDA浓度均下降,差异有统计学意义(P <0.05)。
结论:PC、Heptanol可能通过提高线粒体跨膜电位、减轻线粒体钙超载以致改善缺血再灌注心肌细胞损伤、减轻心肌凋亡;PC可降低线粒体氧自由基水平,而与PC心肌保护机制不同的是,HT不能降低线粒体氧自由基水平。
第三部分缺血后处理、Heptanol预处理对兔心肌缺血/再灌注损伤中细胞膜及线粒体Cx43的影响
目的:探讨PC、Heptanol对兔IRI中细胞膜及线粒体Cx43的影响。
方法:兔80只,建立心肌IRI模型,随机分5组,每组16只:假手术组(Sham组)、缺血再灌注组(IR组)、缺血预处理组(IP组)、缺血后处理组(PC组)和Heptanol预处理(HT组)。应用电镜观察各组闰盘结构变化;应用免疫组化、免疫荧光法检测细胞膜Cx43-GJ分布及含量变化;采用差速离心法提取各组缺血区心肌线粒体蛋白,应用Western blot检测线粒体Cx43蛋白含量。进一步从转录水平采用RT-PCR检测Cx43mRNA表达。
结果:(1)闰盘结构变化与Sham组比较,IR组闰盘及GJ结构不清,可见移位、断裂。与IR组比较,IP、PC和HT组的闰盘病变明显减轻。(2)细胞膜Cx43、GJ与Sham组比较,IR组GJ重构明显,胞膜Cx43含量明显减少(P <0.01)。与IR组比较,IP、PC和HT组的GJ重构明显减轻,并且胞膜Cx43含量增加(P <0.01)。(3)线粒体Cx43蛋白与Sham组比较,IR组线粒体Cx43蛋白显著下降(P <0.01);与IR组比较,PC组、HT组和IP组心肌线粒体Cx43明显升高(P <0.01)。
结论:PC、Heptanol预处理可以减轻IRI引起的闰盘损伤、细胞膜和线粒体Cx43的减少及改善GJ重构,细胞膜与线粒体Cx43可能参与了PC和Heptanol预处理的心肌保护作用。
第四部分线粒体ATP敏感钾通道和线粒体Cx43在缺血后处理和Heptanol预处理相关缺血/再灌注损伤中的作用
目的:探讨线粒体ATP敏感钾通道和线粒体Cx43在PC和Heptanol保护兔IRI中的作用。
方法:兔112只,建立IRI模型,随机分7组,每组16只:假手术组(Sham组)、缺血再灌注组(IR组)、缺血后处理组(PC组)和Heptanol预处理(HT组)、缺血后处理组+5HD组(PC+5HD组)和Heptanol预处理+5HD组(HT+5HD组)。假手术组左冠状动脉前降支近端穿线但不结扎,观察4h,其余4组则结扎30min后,予4h再灌注。取血测定结扎前、结扎后30min、再灌注30min、再灌注2h、再灌注4h血浆CK-MB、cTnI以及SOD与MDA含量。实验结束后,Even’s和TTC染色法测心梗面积。电镜观察心肌细胞超微结构尤其是线粒体与闰盘结构的变化。TUNEL法检测缺血心肌细胞凋亡。免疫荧光检测胞膜cx43-GJ变化以及胞膜Cx43含量,差速离心法提取线粒体,并检测线粒体膜电位、Ca2+浓度、MDA浓度、SOD活性的改变。应用Western Blot检测线粒体Cx43蛋白含量。进一步用RT-PCR检测心肌组织中Cx43mRNA改变。
结果:再灌注4h末,IP、PC和HT组的血浆CK-MB、cTnI及心梗面积明显低于IR、PC+5HD和HT+5HD组(P <0.01)。与IR、PC+5HD和HT+5HD组比较,IP、PC和HT组兔心肌细胞凋亡减少,心肌、闰盘及线粒体形态结构改变明显减轻,线粒体跨膜电位、SOD活性明显升高,线粒体Ca2+浓度、MDA浓度均下降。免疫荧光检测细胞膜Cx43、GJ及Western Blot检测线粒体Cx43蛋白发现,与Sham组比较,IR、PC+5HD和HT+5HD组细胞膜与线粒体Cx43蛋白显著下降(P <0.01);与IR组比较,PC、HT、IP、PC+5HD和HT+5HD组心肌胞膜和线粒体Cx43明显升高(P <0.01和P <0.05))。与PC+5HD和HT+5HD组比较,IP、PC和HT组胞膜Cx43及线粒体Cx43蛋白明显升高(P <0.01)。
结论:PC、Heptanol预处理可以减轻IRI引起的闰盘损伤、改善GJ重构、减轻IRI所致的细胞膜及线粒体Cx43的含量,其可能通过提高线粒体跨膜电位、减轻线粒体钙超载、改善缺血再灌注心肌细胞凋亡等来达到对兔IRI的保护作用。细胞膜、线粒体Cx43可能参与了PC和Heptanol预处理的心肌保护作用,其机制与mitoKATP有关。PC也可降低线粒体氧自由基水平。
Myocardial ischemia reperfusion frequently prone to ischemia-reperfusion injury (IRI), myocardial ischemia-reperfusion injury is a common clinical phenomenon of myocardial injury and its mechanism complex.Myocardial protection interventions currently include ischemic preconditioning (IP), pharmacological preconditioning and ischemic postconditioning (PC) .In view of PC and IP, as well as pharmacological preconditioning, there are some similar protection mechanisms among the treatments mentioned above . Studies find that PC plays a significant role in myocardial protection, but its exact mechanism has not yet to be clarified. The vivo and vitro studies confirmed that dephosphorylation of Cx43, and the opening of gap junction(GJ) channel at the early stage of IRI, which cause the widespread of necrosis agents and aggravate the process of infarction.GJ participate in the process of IRI, take GJ as the target of anti-IRI therapy may be a new direction of development.In 2005, Schulz found the expression of connexin 43(Cx43) in myocardial mitochondrial for the first time, and recently, the role of mitochondrial Cx43 in IP is also one of the hot-top studies. Recent researches find that heart mitochondria and mitochondrial Cx43 play a very important role in myocardial IRI and IP protection. While IP myocardial protection of mitochondrial ATP-sensitive potassium channel (mitoKATP) involved in regulation of GJ.
And Cx43 in PC and heptanol pretreatment has the few studies, particularly the mitochondria Cx43 role in the PC and heptanol preconditioning has not been reported. We assume that mitochondrial Cx43 in PC and heptanol preconditioning also plays an important role. PC and heptanol protection mechanisms may also by activating mitochondrial-sensitive potassium channel,so that there are certain changes in quantities,distribution and function of cell membrane and mitochondrial Cx43,which can prevent the disffusion of injury agents,enhance the stabilization of mitochondrial and anti-injure competence,then attenuate the hazardous effect of IRI. This study are designed to investigate whether the mitochondria and mitochondrial Cx43 are involved in PC and heptanol on myocardial protection and its possible mechanism.
In this study, we built rabbit myocardial IRI model, with PC and Heptanol pretreatment and 5 - hydroxy caproic acid (mitochondrial ATP-sensitive potassium channel specific blocker,5HD) treatment.The protective effect of PC and Heptanol on IRI and their impact on mitochondrial Cx43 will be observed,then explore the possible protective mechanisms of mitochondrial Cx43 in the PC and Heptanol treatment on rabbits with acute myocardial IRI .The topic to be studied is divided into four parts, namely (1)Impact of PC and Heptanol pretreatment on rabbit myocardial IRI; (2) Impact of PC and Heptanol pretreatment on myocardial apoptosis and mitochondrial structure and function in rabbit myocardial IRI; (3) Impact of PC and Heptanol pretreatment on Cx43 of membrane and mitochondria in rabbit myocardial IRI; (4) Impact of PC and Heptanol pretreatment involves mitochondrial ATP-sensitive potassium channel,and the relationship between the expression of mitochondria Cx43 suffered from ischemia-reperfusion .
Part 1 Effection of Ischemic postconditioning and heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits
Objective: To investigate the protection of ischemic postconditioning and Heptanol preconditioning on myocardial isehemia/reperfusion injury of rabbits.
Methods:80 New Zealand white rabbits were randomly divided into five groups, namely sham-operated group (Group Sham), myocardial ischemia / reperfusion model group (Group IR), ischemic preconditioning group (Group IP), ischemia and postconditioning group (PC) and heptanol pretreatment group (Group HT), n = 16. Sham-operated group, thread the proximal left anterior descending(LAD) coronary artery without ligation and observed 4h, while in other groups, ligated LAD for 30min, then released for reperfusion for 4h. Determin the concentration of plasma creatine kinase isoenzyme (CK-MB), troponin (cTnI), SOD and MDA level prior to ligation ,ligation taken 30min, reperfusion 30min, reperfusion 2h and reperfusion 4h. After reperfusion, myocardial infarction areas were measured by Evans blue and TTC staining.SOD and MDA level were measured by prepared homogenate of Myocardial tissue. The SOD and MDA in serum and myocardium were measured respectively.HE stained first,then observed ultrastructural changes of myocodrial by electron microscope.
Results: 1. Enzymatic markers: compared to sham group, the values of CK-MB, cTnI in other groups at the points of ligation 30min, reperfusion 30min, reperfusion after 2h and 4h reperfusion were significantly highe (P <0.01). Compared with the Group IR, the Group IP, PC and HT CK-MB, cTnI values significantly decreased at reperfusion 30min, reperfusion after 2h and 4h reperfusion (P <0.01). 2. Hemodynamics: there was no statistical significance in HR, MAP, LVEDP + dp / dtmax and -dp/dtmax before ischemia among different groups. After reperfusion, compared to Group sham, HR, MAP, + dp/dtmax, -dp/dtmax in treatment groups after ligation 30min, reperfusion 30min, reperfusion 2h, reperfusion 4h decreased, but LVEDP increased significantly (P <0.01);compared to Group IR, MAP, + dp / dtmax, -dp/dtmax (P <0.05) and LVEDP (P <0.01) indexes at the points of reperfusion 2h, 4h significantly improved in IP, PC and HT groups. 3. Infarct area: Group IP (18.97%±2.8%), Group PC (19.07%±4.9%) and the Group HT (20.01%±3.9%) of myocardial infarct areas are significantly smaller than Group IR (35.67%±5.8%,P <0.01). 4. Myocardial structure modification and changes of myocardial ultrastructure: light microscope and electron microscope results, compared to Group sham, there is a significant myocardial injury in Group IR; in contrast of Group IR, IP, PC and HT receive a slighter myocardial cell injury. 5. Myocardium tissue and changes in serum SOD and MDA: 4h after ischemia, compared to Group sham,Group IR SOD activity significantly decreased, while MDA significantly increased(P <0.01). Compared to Group IR, Group IP and Group PC have significantly higher SOD activitives, MDA significantly decreased(P <0.01); there is no significant differences of SOD and MDA in HT group (P >0.05).
Conclusion: Ischemic postconditioning and Heptanol pretconditioning can protect the heart from IRI injury. But Heptanol preconditioning has no effect on SOD activity and MDA content. This is the difference from the Ischemic postconditioning about pretecting IRI injury.
Part 2 Effect of ischemic postconditioning and Heptanol on apoptosis and structural changes of mitochondria in ischemia reperfusion injury of rabbit
Objective:To investigate the effects of ischemic postconditioning and Heptanol preconditioning on apoptosis and structural and functional changes of mitochondria induced by IRI of rabbits in vivo, in order to discuss the mechanisms of ischemic postconditioning and Heptanol preconditioning from the view of mitochnodial.
Methods: Eighty rabbits were divided randomly into five groups:sham operation group(Group Sham),ischemic reperfusion group (Group IR),ischemic preconditioning group (Group IP), ischemic postconditioning group (Group PC) and Heptanol preconditioning group (Group HT) ,with sixteen rabbits in each group.All rabbits were sacrificed after reperfusion for 4h.The heart was quickly removed for observing the structure of mitochondria and measurement of the apoptosis fraction by TUNEL. We observed ultrastructural changes of myocardium under electron microscope and examined mitochondrial membrane potential and Ca2+ concentration, MDA content and SOD activity of myocardial mitochondria.
Results: (1)The results of cardiac muscle cell apoptosis index:TUNEL positive cells obviously increased in Group IR, and with an intensive distribution. The AI of five groups were respectively Group sham(0.12%±0.07% ), Group IR(33.2%±1.2%),Group IP(14.4%±2.3%), Group PC(15.6%±1.9%)and Group HT(15.8%±1.8%) .The TUNEL positive cells of Group IP,PC and HT are obviously less than Group IR and the difference was significant(P <0.01). (2) The changes of mitoehondrial ultrastrueture: Compared to Group IR, in Group IP , Group PC and Group HT, the damages of mitoehondrial ultrastrueture were milder(P <0.01).(3) The results of mitochondrial function in five groups: Compared to Group IR, in Group IP, Group PC and Group HT, mitochondrial membrane potential(P <0.01) was significantly higher and Ca2+ concentration were much lower(P <0.05).No significant difference was found in indicators between Group IP , Group PC and Group HT. Compared to Group IR, MDA content and SOD activity of myocardial mitochondria in Group HT, no significant difference was found;But in Group IP and Group PC, MDA content were much lower and SOD activity was significantly higher((P <0.05).
Conclusion: Ischemic postconditioning and Heptanol preconditioning can protect the heart from IRI evidenced by rehabilitating mitochondrial ultrastructure,preventing apoptosis, increaseing mitochondrial membrane potential and alleviating Ca2+ overload in myocardial mitochondria. Be different from HT, PC can reduce the levels of oxygen free radicals in mitochondrial. This is the difference between PC and HT in myocardial protection.
Part 3 Impact of Ischemic postconditioning and heptanol preconditioning on Cx43 of membrane and mitochondria in rabbit myocardial ischemia-reperfusion injury
Objective: To investigate the effects of ischemic postconditioning and Heptanol preconditioning on Cx43 of membrane and mitochondria in rabbit myocardial ischemia-reperfusion injury
Methods: 80 New Zealand white rabbits were randomly divided into five groups, namely sham-operated group (Group Sham), myocardial ischemia / reperfusion model group (Group IR), ischemic preconditioning group (Group IP), ischemia postconditioning group (Group PC) and heptanol pretreatment group (Group HT), n = 16. All rabbits were sacrificed after reperfusion for 4h. Application of electron microscopy to illustrate the modification of intercalated disk; application of immunohistochemistry and immunofluorescence staining to observe the changes of membrane distribution and the content of Cx43; mitochondrial protein was extracted using gradient centrifugation. The mitochondria Cx43 were detected by Western Blotting.Further detection Cx43mRNA expression from the transcription level by Reverse Transcription Polymerase Chain Reaction.
Results:Compared to Group IR, there were moderate disorganized GJ distribution in Group IP , Group PC and Group HT. Compared to Group Sham, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly decreased in Group IR (P <0.01); These results were further confirmed by Reverse Transcription–Polymerase Chain Reaction.No significant difference was found among Group IP ,Group PC and Group HT(P >0.05).
Conclusion: Ischemic postconditioning and Heptanol preconditioning can protect the heart from ischemia reperfusion injury, it can rehabilitate the injured intercalated disc, remodel its reconstruction and prevent Cx43 decreases induced by IRI both in membrane and mitochondria. Myocardial protection of PC and heptanol preconditioning may involve Membrane and mitochondrial Cx43.
Part 4
Impact of PC and heptanol pretreatment involved in mitochondrial ATP-sensitive potassium channel on Cx43 of mitochondria in rabbit ischemia-reperfusion injury
Objective: To investigate Impact of PC and heptanol pretreatment involved in mitochondrial ATP-sensitive potassium channel on mitochondria Cx43 in rabbit ischemia-reperfusion injury.
Methods: 112 Rabbits, established myocardial ischemia-reperfusion model, were randomly divided into seven groups: sham operation group (Group Sham), ischemia reperfusion group (Group IR), ischemic postconditioning group (Group PC), and Heptanol preconditioning +5HD group (Group PC +5HD) and heptanol pretreatment +5HD group (Group HT +5HD), n=16. Sham-operated group, thread the proximal left anterior descending coronary artery without ligation and observed for 4h, while the other six groups, after ligation for 30min, released the ligature, reperfused for 4h. Determined the concentration of carotid artery of plasma creatine kinase isoenzyme (CK-MB), troponin (cTnI), as well as SOD and MDA prior to ligation ,ligation for 30min, reperfusion for 30min, reperfusion for 2h and reperfusion for 4h. After reperfusion, myocardial infarction areas were measured by Evans blue and TTC staining, myocardial structure and ultrastructure were observed by HE staining and electron microscopy and especially focus on mitochondria and intercalated disc structure.TUNEL assayed apoptosis in ischemic myocardium. Immunofluorescence detected membrane changes in GJ and Cx43 membrane content. Extracted mitochondria by gradient centrifugation and detected mitochondrial membrane potential, Ca2+ concentration, malondialdehyde(MDA) concentration, superoxide dismutase (SOD) activity of myocardial mitochondria.Analyze the content of mitochondrial Cx43 protein by Western blotting and further detection Cx43mRNA expression from the transcription level by Reverse Transcription Polymerase Chain Reaction.
Results: (1) Plasma CK-MB and cTnI activity and Myocardial infarct areas: After IR 4h, compared to Group IR,Group PC+5HD and Group HT+5HD,the CK-MB,cTnI and myocardial infarct areas of IP, PC and HT were obviously smaller(P <0.01). (2) The results of myocardium cell apoptosis index: compared to Group IR, Group PC+5HD and Group HT+5HD,TUNEL positive cells obviously decreased in IP, PC and HT myocardium tissue (P <0.01). (3) The modification of myocardial ultrastructure, GJ and mitochondrial: compared to Group IR, Group PC+5HD and Group HT+5HD,the modification of myocardial ultrastructure, GJ and mitoehondrial of IP, PC and HT were milder(P <0.01). (4) The results of mitochondrial function in five groups: Compared to Group IR, Group PC+5HD and Group HT+5HD, in IP,PC and HT, mitochondrial membrane potential was significantly higher and Ca2+ concentration were much lower(P<0.01).No significant difference was found among IP , PC and HT groups. Compared to Group IR, there was no significant difference in MDA content and SOD activity of myocardial mitochondria in Group HT;But in Group IP and Group PC, MDA content were much lower and SOD activity was significantly higher(P <0.05).(5) Compared to Group IR, Group PC+5HD and Group HT+5HD,the disorganized gap junction distribution in IP , PC and HT groups were milder (P <0.01). Compared to Group Sham, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly decreased in IR, PC+5HD and HT+5HD groups(P <0.01); Compared to Group IR, the sarcolemma Cx43 and the mitochondria Cx43 expression distinctly increased in IP,PC and HT groups (P <0.01) and PC+5HD and HT+5HD groups (P <0.05) . Compared to PC+5HD and HT+5HD groups, the sarcolemma Cx43-GJ and the mitochondria Cx43 expression obviously increased in IP,PC and HT groups (P <0.01).No significant difference was found among IP , PC and HT groups (P >0.05).
Conclusion: PC, Heptanol pretreatment can reduce the intercalated discs injury caused by IRI, improving GJ reconstruction, to reduce IRI induced degradation of Cx43 cell membrane and mitochondria, which may be by increasing mitochondrial membrane potential, reducing mitochondrial calcium overload, improving myocardial apoptosis and protection of myocardial mitochondrial structure of rabbit IRI. Mitochondrial Cx43 may be involved in the PC and Heptanol preconditioning on myocardial protection, its mechanism related to mitochondrial ATP-sensitive potassium channel. PC can also reduce the level of oxygen free radicals.
引文
[1] Murry CE, Jennings RB, Reimer KA. Preeonditioning with ischemia: adelay of lethal cell injury in ischemic myoeardium. Circulation,1986,74:1124 -1136
[2] Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol,2003,285:H579-H588.
[3] Min Zhu, Jianhua Feng, Eliana Lucchinetti, et al. Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvage kinase pathway, cardiovascular research,2006,72:152-62.
[4] Lisa M. Schwartz and Claudia J. Lagranha,et al. Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs. Am J Physiol Heart Circ Physiol, 2006, 290:H1011- H1018.
[5] Patrick Staat, Gilles Rioufol, Christophe Piot, et al. Postconditioning the Human Heart, Circulation, 2005,112:2143-2148.
[6] Quist AP, Rhee SK, Lin H, et al. Physiological role of gap junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol,2000, 148: 1063–1074.
[7] Garcia-Dorado D, Vinten-Johansen J, Piper HM. Bringing preconditioning and postconditioning into focus. Cardiovascular Research, 2006, 70(2): 167-169.
[8] De MA, Vega VL, Contreras JE. Gap junctions, homeostasis, and injury. J Cell Physiol,2002, 191(3):269–282.
[9] Quist AP,Rhee SK,Lin H,et al.Physiological role of gap junctional hemichannels. Extracellular calcium dependent isosmotic volume regulation. J Cell Bio, 2000, 148: 1063-1074.
[10] Kang J, Kang N, Lovatt D, et al. Connexin 43 hemichannels are permeable to ATP. J Neurosci,2008,28:4702-4711.
[11] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res, 2005, 97(6):583-586.
[12] Saltman AE,Aksehirli TO,Valiunas V,et al.Gap junction uncoupling protectsthe heartagainst ischemia.J Thorac Cardiovasc Surg,2002,124:371-376.
[13] Naitoh K, Ichikawa Y, Miura T, et al. MitoKATP channel activation suppresses gap junction permeability in the ischemic myocardium by an ERK-dependent mechanism. Cardiovascular Research, 2006,70:374-383.
[14] Schwanke U,Konietzka I,Duschin A,et al.No ischemic preconditioning in heterozygousconnexin43-deficient mice.Am J Physiol Heart Circ Physiol,2002, 283:1740-1742.
[15] Ruiz-Meana M, Rodríguez-Sinovas A, et al. Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury.Cardiovasc Res,2008,77:325-333.
[16] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol,2007,292: H1764-H1769.
[17] Goubaeva F, Mikami M, Giardina S, et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun, 2007,352: 97-103.
[18] Dhein S. New, emerging roles for cardiac connexins: Mitochondrial Cx43 raises new questions. Cardiovasc Res, 2005,67:179-181.
[19] Boengler K, Dodoni G, Rodriguez-Sinovas A, et al. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res,2005,67: 234–244.
[20] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res, 2005, 97:583-586.
[21] Heusch G, Büchert A, Feldhaus S, et al. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol,2006,101:354–356.
[22] Boengler K, Buechert A, Heinen Y, et al. Cardioprotection by ischemic postconditioning is lost in aged and STAT3-deficient mice. Cir Res,2008,102: 131-135.
[23] Kerstin Boengler, Ina Konietzka, Astrid Buechert, et al. Loss of ischemic preconditioning’s cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43, Am J Physiol Heart Circ Physiol, 2007, 292: H1764–H1769.
[24] Garcia-Dorado D, Vinten-Johansen J, Piper HM. Bringing preconditioning andpostconditioning into focus. Cardiovascular Research,2006,70: 167-169.
[25] Huffmyer J, Raphael J. Physiology and pharmacology of myocardial preconditioning and postconditioning. Semin Cardiothorac Vasc Anesth,2009,13:5-18.
[26] Lamendola P, Di Monaco A, Barone L,et al. Mechanisms of myocardial cell protection from ischemia/reperfusion injury and potential clinical implications,G Ital Cardiol (Rome),2009,10:28-36.
[27] Schulz R, Boengler K, Totzeck A,et al. Connexin 43 in ischemic pre- and postconditioning. Heart Fail Rev,2007,12:261-266.
[1] Murry CE, Jennings RB, Reimer KA. Preeonditioning with ischemia: adelay of lethal cell injury in ischemic myoeardium.Circulation,1986,74:1124-1136
[2] Cruciani V, Mikalsen S-O. Connexins, gap junctional intercellular communication and kinases. Biol Cell,2002,94:433-443.
[3] David GD,Javier Inserte,Marisol RM,et al.Gap junction uncouples heptanol prevents cell-to-cell pregression of hypercontracture and limits necrosis during myocardial reperfusion.Circulation,1997,96: 3579-3584.
[4] Masaharu A,Hideo 0,Minoru H,et al.Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.J Clin Invest,1997,100:3053-3059.
[5] Yellon DM, Baxter GF. Protecting the ischemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality? Heart,2000,83:381-387.
[6] Kloner RA,Arimie RB,Kay GL,et al.Evidence for stunned myocardium in humans: a 2001 update.Coron Artery Dis,2001,12:349-356.
[7] Bolli R.The late phase of preconditioning.Circ Res,2000,87:972-983.
[8] Zhao ZQ, Corvera JS, Halkos ME,et al.Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparision with ischemic preconditioning. Am J Physio1,2003,285:579-588.
[9] Zhu M, Feng JH, Lucchinetti E,et al.Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvagekinase path way.cardiovascular research,2006,72:152-162.
[10]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[11] Jenings RB, Sommers HM, Smyth GA,et al.Myocardial necrosis induced by tempornary ocllusion of a coronary artery in the dog.Arch Pathol,1960,70:68-78.
[12]徐清斌,张馥敏,杨志健,等.局部注射自体骨髓间充质干细胞修复心梗后心肌的实验研究.江苏医药杂志,2004,30:266-267.
[13] Fung K,Weisel RD. Enhanced myocardial angiogenesis by gene transfer with transplanted cells.Circulation,2001,104:1218-1222.
[14]陈运贞.慢性心肌梗塞大鼠实验模型.重庆医科大学学报,2002,27:153-155.
[15]徐美清,何华等.犬急性心肌梗死模型制备的方法学研究.安徽医学,2002,23:6-8.
[16]杨国勋,刘唐威,钟国强,等.心肌梗死诊断中的应用.广西医学,2007,29:1135-1138.
[17] Tsang XL,Sato H,Tiwari S,et al.Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions <45 min. Am J Physiol Heart Circ Physiol,2006, 291:H2308-H2317.
[18] Daring CE,Jiang R,Marnard M,et al.postcondition via stuttering reperfusion limits myocardial infact size in rabbit hearts role of ERK1/2.Am J Physol Heart Circ Physiol,2005,289:H1618-1626.
[19] David GD.Myocardial reperfusion injury:a new review.Cardiaovasc Res, 2004, 61:363-364.
[20] Galinnanes M, Fowler AG.Role of clinical pathologies in myocardial injury following ischemia and reperfusion. Cardiaovasc Res,2004,61:512- 521.
[21] Kato R,Foex P.Myocardial protection by anesthetic agents against ischemia-reperfusion injury:an update for anesthesiologists.Can J Anaesth, 2002,49:777-791.
[22] Rong F,Peng Z,Ye MX,et al.Myocardial apoptosis and infarction after ischemia/reperfusion are attenuated by kappa-opioid receptor agonist. Arch Med Res,2009, 40:227-234.
[23] Piao CS, Gao S, Lee GH, et al. Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels. Pharmacol Res, 2010,61:342-348.
[24] Gaugron P, Eilles C, Kugler L, et al. Progressive left ventricular dysfunction and remodeling after myocardial infarction potential mechanisms and early predictiors.Circulation,1993,87:755-763.
[25] Bolognese L,Ducci K,Angioli P,et al. Elevations in troponin I after percutaneous coronary interventions are associated with abnormal tissue level perfusion in high risk patients with non ST segment elevation acute oronary syndromes. Circulation,2004, 110:1592-1597.
[26] Zaugg M,Lucchinetti E,Garcia C,et al.Anaesthetics and cardiac precondition. Clinical implications.Br J Anaesth,2003,91:566-576.
[27] Philipp S, Downey M, Cohen MV,et al.Postconditioning must be initiated in less than 1 min following reperfusion and is dependent on adenosine receptors and PI3-kinase. Circulation,2004,110-l68.
[28] Ohtake M,Morino S,Kaidoh T,et,al.Three dimensional structural changes in cerebral microveasels after transient focal cerebral ischemia in rats scanning electron micoscopic study of corrosion casts.Neuropa theology,2004,24:219-227.
[29] Zatta AJ,Kin H,Lee G,et,al.Infarct sparing effect of myocardial postconditioning is dependent on protein kinase C signalling.Cardiovasc Res,2006,70:315-324.
[30] Li CM, Zhang XH, Ma XJ, et al. hearts with acute myocardial Impact of remote postconditioning on rabbit ischemic reperfusion injury.Chin J of Patho physiology,2006, 22:2332-2335.
[1] Milerova M,Charvatova Z,Skarka L,et al.Neonatal cardiac mitochondria and ischemia/reperfusion injury. Mol Cell Biochem. 2010,335:147-153.
[2] Honda HM,Korge P,Weiss JN. Mitochondria and ischemia reperfusion injury.Ann N Y Acad Sci,2005,1047:248-258.
[3] Maulik N,Yoshida T,Engelman RM,et al.Ischemic-preconditioning attenuates apoptotic cell death as sociated with ischemia/reperfusion.Mol Cell Biochem 1998,186:139-145
[4] Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol,2003,285:H579-H588.
[5] Zhu M, Feng JH, Lucchinetti E,et al.Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvagekinase path way.cardiovascular research,2006,72:152-162.
[6] Schulz R,Boengler K,Dodoni G,Ruiz-Meana M,et al.Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.Cardiovasc Res,2005,67:234-244.
[7]曾玉杰,冯义柏,于世龙,等.卡维地洛和庚醇对心肌缺血再灌注损伤的保护作用.中华老年多器官疾病杂志,2007,6:123-127.
[8] Gavrieli Y,Sherman Y,Ben-Sasson SA.Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation.J cell Biol, 1992,119:493-501.
[9] Flameng W, Borgers M, Daenen W,et al. Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J Thorac Cardiovasc Surg,1980,79:413-424.
[10] Emaus RK,Grunwald R,Lemaslers JJ.Rhodamine 123 as a probe of Iransmem brane potential in isolated rat liver mitochondria:spectra1 and metabolic properties.Biochim Biophys Acta,1986,850:436-448.
[11] Elizabeth AF,James DM,Takttro T,et al.Myocardial mitochondrial calcium accumulation modulates nuclear calcium accumulation and DNA Fragmentation.Amm Thorac Surg,1995,60:338-334.
[12]司徒镇强,吴军正,主编.细胞培养.第1版.西安:世界图书出版西公司,1996. 213-214.
[13] Machado NG, Alves MG, Carvalho RA, et al. Mitochondrial involvement in cardiac apoptosis during ischemia and reperfusion: can we close the box? Cardiovasc Toxicol,2009,9:211-227.
[14] Fliss H. Accelerated apoptosis in friend of foe. Basic Res Cardiol reperfusion myocardium,1998,93:90-96
[15] Zhang LP, Ma HJ, Bu HM, et al. Polydatin attenuates ischemia/reperfusion-induced apoptosis in myocardium of the rat.Sheng Li Xue Bao,2009,61:367-372.
[16] Castedo E,Segovia J,Escudero C,et al.Ischemia-reperfusion injury during experimental heart transplantation. Rev Esp Csrdiol,2005,58: 941-950.
[17] Gross GJ,Kersten JR,Warltier DC.Mechanisms of postischemic contractile dysfunction.Thorac Surg,1999,68:1898-904.
[18] Baumgartner HK,Gerasimenko JV,Thorne C,et al. Calcium elevation in mitochondria is the main Ca2+ requirement for mPTP opening. J Biol Chem, 2009,284:20796-20803.
[19] Novgorodov SA, Gudz TI.Ceramide and mitochondria in ischemia/reperfusion. J Cardiovasc Pharmacol,2009,53:198-208.
[20] Mattson MP,Kroeme G.Mitochondria in cell death:novel targets for neuroprotectionand cardioprotection.Trends Mol Med,2003,9:196-205.
[21] Arrell DK,Elliott ST, Kane LA,et al. Proteomic analysis of preconditioning: novel protein targets converge to mitochondria metabolism pathways.Circ Res,2006,99:706-714.
[22]吴青,陶红凯等.缺血再灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究.中国心血管病研究杂志,2004,2:905-908.
[23]徐少东,马礼坤,屈朝法.犬急性心肌梗死晚期再灌注对心肌Fas/FasL系统的影响及其可能的氧化应激机制.中国病理生理杂志2007,23:307-310.
[24] Ratha J, Majumdar KN, Dhara K, et al. Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase dependent and independent pathways. Mol Cell Biochem, 2007,304:143-154.
[25] Zanna C,Ghelli A,Porcelli AM,et al.Caspase independent death of Leber’s hereditary optic neuropathy cybrids is driven by energe tic failure and mediated by AIF and endonuclease G.Apoptosis,2005,10:997-1007.
[26] Li XZ,Liu JX.Role of autophagy in cardiac myocyte during ischemia/reperfusion.Chinese Pharmacological Bulletin,2008,24:704-707.
[27]杨兴易,石正蒙,顾桂国等.参附注射液对心肺复苏后大鼠心肌细胞保护作用的研究.临床急诊杂志,2005,6:16-19.
[28] Paradies G,Petrosillo G,Pistolese M,et a1.Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart:involvement of reactive oxygen species and cardiolipin.Circ Res,2004,94:53-59.
[1] Schulz R, Heusch G1.Connexin43 and ischemic preconditioning.Adv Cardiol,2006,42:213-227.
[2] Solan JL, Lampe PD. Connexin43 phosphorylation: structural changes and biological effects. Biochem J,2009419:261-272.
[3]辛莉,郭国庆,沈伟哉.间隙连接蛋白家族的研究进展.中国病理生理杂志, 2007,23: 1240-1243.
[4] Boengler K,Dodoni G,Rodriguez-Sinovas A,et al.Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic precondi tioning.Cardiovasc Res,2005,67:234-244.
[5] Goubaeva F, Mikami M,Giardina S,et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun,2007,352:97-103.
[6] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006,99:93-101.
[7] Saltman AE,Aksehirli TO,Valiunas V,et al.Gap junction uncoupling protects the heart against ischemia.J Thorac Cardiovase Surg,2002,124:371-376.
[8]陈宝平,夏强,沈岳良.缝隙连接失耦剂Heptanol对缺血心肌的影响,中国心脏起搏与心电生理杂志,2002,16:139-142.
[9] Carcia- Dorado D, Theroux P, Desco M, et al. Cell-to-cell interaction:amechanism to explain wave-front progression of myocardial necrosis. Am physio soc, 1989,214: H1266-1273.
[10]苏德淳,常志文,范书英.缝隙连接在大鼠缺血预适应心肌保护中的作用.中华心血管病杂志,2006,34:690-694.
[11] Schwanke U,Konietzka I,Duschin A, et al. No ischemic preconditioning in heterozygous connexin 43 deficient mice.Am J Physiol Heart Cire Physiol, 2002,283:1740-1742.
[12] Daleau P, Boudriau S, Michaud M, et al. Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and Pharmacol ,2001,79:371-378.
[13] Quist AP, Rhee SK, Lin H, et al. Physiological role of gap functional hemichannels extracellular calcium-dependent volume regulation. J Cell Biol,2000,148:1063-1074.
[14]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[15] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006, 99: 93–101.
[16] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol, 2007, 292: H1764-H1769.
[17] Schwanke U, Konietzka I, Duschin A, et al. No ischemic preconditioning in heterozygous connexin43-deficient mice. Am J Physiol Heart Circ Physiol,2002, 283: H1740–H1742.
[18] Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases.Biol Cell,2002,94:433–443.
[19] Evans WH, Martin PE. Gap junctions:Structure and function.Mol Membr Biol, 2002,19:121-125.
[20] Jeyaraman M,Tanguy S,Fandrich RR,et al.Ischemia-induced dephosphorylation of cardiomyocyte connexin 43 is reduced by okadaie acid and calyculin A but not fostriecin.Mol Cell Biochem,2003,242:129-134.
[21] De MA,Vega VL,Contreras JE. Gap junctions,homeostasis,and injury. J Cell Physiol,2002,191:269–282.
[22] Goubaeva F,Mikami M,Giardina S,et al.Cardiac mitochondrial connexin 43 regulates apoptosis.Biochemical and Biophysical Research Communications,2007,352:97-103.
[23] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res,2005,97:583-586.
[24] Boengler K, Konietzka I , Buechert A,et al. Loss of ischemic preconditioning cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43.Am J Physiol Heart Circ Physio1,2007,292:H1764-H1769.
[25] Boengler K,Dodoni G,Rodriguez-Sinovas A,et al.Connexin 43 in cardiomyocytemitochondria and its increase by ischemic precondi tioning.Cardiovasc Res,2005,67:234-244.
[26] Boengler K, Ruiz-Meana M, Miro-Casas B, et al.Connexin 43 controls mitochondrial respiration. Circulation ,2006, 114:243-251.
[27] Miro-Casas R, Ruiz-Meana M, Agullo R, et al. Connexin43 in the inner mitochondrial membrane of cardiomyocytes forms hemichannels that contribute to matrix volume regulation. Circulation,2007,116-167.
[28] Saffitz JE,Laing JG,Yamada KA,et a1.Connexin expression and turnover :implications for cardiac excitability.Circ Res,2000,86:723-728.
[29] Fernandez-Cobo M,Gingalewski C,Dmjan D.Down regrudation of connexin 43 gene expression in rat heart during inflammation:The role of tumor necrosis factor.Cytokine,l999,ll:216-224.
[30] Solan JL,Lampe PD.Connexin43 phosphorylation: structural changes and biological effects.Biochem J,2009,419:261-272.
[1] Naitoh K, Ichikawa Y, Miura T,et al.MitoKATP channel activation suppresses gap junction permeability in the ischemic myocardium by an ERK-dependent mechanism. Cardiovasc Res,2006,70:374-83.
[2] Mykytenko J, Reeves JG, Kin H, et al.Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K(ATP) channels during reperfusion. Basic Res Cardiol,2008,103:472-484.
[3] O'Rourke B.Evidence for mitochondrial K+ channels and their role in cardioprotection.Circ Res,2004,94:420-432.
[4] Kamada N,Kanaya N,Hirata N,et al. Cardioprotective effects of propofol in isolated ischemia-reperfused guinea pig hearts: role of KATP channels and GSK-3beta. Can J Anaesth,2008,55:595-605.
[5] Simerabet M, Robin E, Aristi I, et al.Preconditioning by an in situ administration of hydrogen peroxide: involvement of reactive oxygen species and mitochondrial ATP-dependent potassium channel in a cerebral ischemia-reperfusion model. Brain Res,2008,1240:177-184.
[6] Korge P,Honda HM,Weiss JN.K+ -dependent regulation of matrix volume improves mitachondrial function under conditions mimicking ischemia reperfusion.Am J Physiol Heart Circ Physiol,2005,289:H66-H77.
[7] Yang XM,Proctor JB,Cui L,et al.Multiple,brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways . Am Coil Cardiol,2004,44:l103-1110.
[8] Dhein S. Gap ,junction channels in the cardiovascular system: pharmacological And physiological modulation.TrendsPharmacol Sci,1998,19:229-241.
[9] Kevelaitis P,Peynet J,Mouas C,et al.Opening of potassium channels:The common cardioprotective link between preconditioning and natural hibernation? Circulation,1999,99:3079-3085.
[10] Quist AP,Rhee SK,Lin H,et al.Physiological role of gap ,functional Hemichannels.Bxtracellular calcium-dependentisosmotic volume regulation.JCell Biol,2000,148:1063-1074.
[11] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 tothe inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006, 99: 93–101.
[12] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res,2006,99:93-101.
[13] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol, 2007, 292: H1764-H1769.
[14] Heinzel FR, Luo Y, Li X, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res,2005,97:583-586.
[15] Penna C, Mancardi D,Raimondo S, et al. The paradigm of postconditioning to protect the heart.Cell Mol Med,2008,12: 435–458.
[16]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[17] Wojtovich AP, Burwell LS, Sherman TA, et al. The C. elegans mitochondrial K+(ATP) channel: a potential target for preconditioning. Biochem Biophys Res Commun,2008,376:625-628.
[18] Mykytenko J,Reeves JG,Kin H,et al.Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K(ATP) channels during reperfusion.Basic Res Cardiol,2008,103:472-484.
[19] Krolikowski JG, Bienengraeber M,Weihrauch D,et al. Inhibition of mitochondrial permeability transition enhances isoflurane-induced cardioprotection during early reperfusion:the role of mitochondrial KATP channels.Anesth Analg,2005,101: 1590-1596.
[20] Quinlan CL, Costa AD, Costa CL, et al. Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels. Am J Physiol Heart Circ Physiol,2008,295:H953-H961.
[1]辛莉,郭国庆,沈伟哉.间隙连接蛋白家族的研究进展.中国病理生理杂志,2007: 1240-1243.
[2] Schulz R, Heusch G.Connexin 43 and ischemic preconditioning.Cardiovasc Res, 2004, 62: 335–344.
[3] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.Circ Res,2006,99: 93–101.
[4] Boengler K, Dodoni G, Rodriguez-Sinovas A, et al. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.CardiovascRes,2005,67:234–244.
[5] Ruiz-Meana M, Rodríguez-Sinovas A, Cabestrero A,et al.Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischemic reperfusion injury.Cardio vascular Research,2008,77:325-333.
[6] Palmer JW, Tandler B, Hoppel C, et al. Biochemical properties of subsarcolemmal and inter-fibrillar mitochondria isolated from rat cardiac-muscle. J Biol Chem,1977,252: 8731–8739.
[7] Boengler K, Stahlhofen S, van de Sand A, et al. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria. Basic Res Cardiol, 2009, 104: 141-147.
[8] Li H, Brodsky S, Kumari S, et al. Paradoxical overexpression and translocation of connexin 43 in homocysteine-treated endothelial cells. Am J Physiol Heart Circ Physiol, 2002, 282: H2124–H2133.
[9] Schulz R, Boengler K, Totzeck A, et al. Connexin 43 in ischemic pre- and postconditioning. Heart Fail Rev, 2007, 12: 261–266.
[10] Totzeck A, Boengler K, van de Sand A, et al. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning[J]. Am J Physiol Heart Circ Physiol, 2008, 295(5): H2106-2012.
[11] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43.Am J Physiol Heart Circ Physiol,2007,292: H1764-H1769.
[12] Schulz R, Gres P, Skyschally A,et al. Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo. FASEB J,2003,17: 1355–1357.
[13] Dupont E, Matsushita T, Kaba RA, et al. Altered connexin expression in human congestive heart failure. J Mol Cell Cardiol, 2001, 33: 359–371.
[14] Doble BW, Dang X, Ping P, et al. Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell– cell contact forming cardiomyocytes. J Cell Sci, 2004,117: 507– 514.
[15]罗红鹤,雷艺炎,陈振光,等.缺血预处理在心瓣膜置换术中对心电活动及细胞缝隙连接的保护作用.中国病理生理杂志,2008,24:1953-1956.
[16] Schwanke U, Konietzka I, Duschin A, et al. No ischemic preconditioning in heterozygous connexin43-deficient mice.Am J Physiol Heart Circ Physiol,2002,283: H1740–H1742.
[17] Heinzel FR, Luo Y, Li X, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res, 2005, 97: 583–586.
[18] Gutierrez PL. The role of NAD(P)H oxireductase (DT-diaphorase) in the bioactivation of quinone-containing antitumor agents: a review.Free Radic Biol Med, 2005, 29: 263–275.
[19] Heusch G, Büchert A, Feldhaus S, et al. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol, 2006, 101:354–356.
[20] Penna C, Mancardi D,Raimondo S, et al. The paradigm of postconditioning to protect the heart . Cell Mol Med, 2008, 12: 435–458.
[21]何燕,曾志羽,钟国强,等.线粒体Cx43参与缺血后处理对兔急性心肌缺血再灌注损伤保护作用.中华心血管病杂志, 2010,38:357-362.
[22]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[23]何燕,曾志羽,钟国强,等.线粒体Cx43参与Heptanol预处理对兔急性心肌缺血再灌注损伤保护作用.中国药理学通报, 2009,25:1660-1665.
[24] Miro-Casas E, Ruiz-Meana M, Agullo E, et al. Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake.Cardiovasc Res,2009,83: 747-756.
[25] Goubaeva F, Mikami M, Giardina S, et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun, 2007, 352: 97-103.
[2] Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol,2003,285:H579-H588.
[3] Min Zhu, Jianhua Feng, Eliana Lucchinetti, et al. Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvage kinase pathway, cardiovascular research,2006,72:152-62.
[4] Lisa M. Schwartz and Claudia J. Lagranha,et al. Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs. Am J Physiol Heart Circ Physiol, 2006, 290:H1011- H1018.
[5] Patrick Staat, Gilles Rioufol, Christophe Piot, et al. Postconditioning the Human Heart, Circulation, 2005,112:2143-2148.
[6] Quist AP, Rhee SK, Lin H, et al. Physiological role of gap junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation. J Cell Biol,2000, 148: 1063–1074.
[7] Garcia-Dorado D, Vinten-Johansen J, Piper HM. Bringing preconditioning and postconditioning into focus. Cardiovascular Research, 2006, 70(2): 167-169.
[8] De MA, Vega VL, Contreras JE. Gap junctions, homeostasis, and injury. J Cell Physiol,2002, 191(3):269–282.
[9] Quist AP,Rhee SK,Lin H,et al.Physiological role of gap junctional hemichannels. Extracellular calcium dependent isosmotic volume regulation. J Cell Bio, 2000, 148: 1063-1074.
[10] Kang J, Kang N, Lovatt D, et al. Connexin 43 hemichannels are permeable to ATP. J Neurosci,2008,28:4702-4711.
[11] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res, 2005, 97(6):583-586.
[12] Saltman AE,Aksehirli TO,Valiunas V,et al.Gap junction uncoupling protectsthe heartagainst ischemia.J Thorac Cardiovasc Surg,2002,124:371-376.
[13] Naitoh K, Ichikawa Y, Miura T, et al. MitoKATP channel activation suppresses gap junction permeability in the ischemic myocardium by an ERK-dependent mechanism. Cardiovascular Research, 2006,70:374-383.
[14] Schwanke U,Konietzka I,Duschin A,et al.No ischemic preconditioning in heterozygousconnexin43-deficient mice.Am J Physiol Heart Circ Physiol,2002, 283:1740-1742.
[15] Ruiz-Meana M, Rodríguez-Sinovas A, et al. Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia-reperfusion injury.Cardiovasc Res,2008,77:325-333.
[16] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol,2007,292: H1764-H1769.
[17] Goubaeva F, Mikami M, Giardina S, et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun, 2007,352: 97-103.
[18] Dhein S. New, emerging roles for cardiac connexins: Mitochondrial Cx43 raises new questions. Cardiovasc Res, 2005,67:179-181.
[19] Boengler K, Dodoni G, Rodriguez-Sinovas A, et al. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res,2005,67: 234–244.
[20] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res, 2005, 97:583-586.
[21] Heusch G, Büchert A, Feldhaus S, et al. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol,2006,101:354–356.
[22] Boengler K, Buechert A, Heinen Y, et al. Cardioprotection by ischemic postconditioning is lost in aged and STAT3-deficient mice. Cir Res,2008,102: 131-135.
[23] Kerstin Boengler, Ina Konietzka, Astrid Buechert, et al. Loss of ischemic preconditioning’s cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43, Am J Physiol Heart Circ Physiol, 2007, 292: H1764–H1769.
[24] Garcia-Dorado D, Vinten-Johansen J, Piper HM. Bringing preconditioning andpostconditioning into focus. Cardiovascular Research,2006,70: 167-169.
[25] Huffmyer J, Raphael J. Physiology and pharmacology of myocardial preconditioning and postconditioning. Semin Cardiothorac Vasc Anesth,2009,13:5-18.
[26] Lamendola P, Di Monaco A, Barone L,et al. Mechanisms of myocardial cell protection from ischemia/reperfusion injury and potential clinical implications,G Ital Cardiol (Rome),2009,10:28-36.
[27] Schulz R, Boengler K, Totzeck A,et al. Connexin 43 in ischemic pre- and postconditioning. Heart Fail Rev,2007,12:261-266.
[1] Murry CE, Jennings RB, Reimer KA. Preeonditioning with ischemia: adelay of lethal cell injury in ischemic myoeardium.Circulation,1986,74:1124-1136
[2] Cruciani V, Mikalsen S-O. Connexins, gap junctional intercellular communication and kinases. Biol Cell,2002,94:433-443.
[3] David GD,Javier Inserte,Marisol RM,et al.Gap junction uncouples heptanol prevents cell-to-cell pregression of hypercontracture and limits necrosis during myocardial reperfusion.Circulation,1997,96: 3579-3584.
[4] Masaharu A,Hideo 0,Minoru H,et al.Myocardial ischemia induces differential regulation of KATP channel gene expression in rat hearts.J Clin Invest,1997,100:3053-3059.
[5] Yellon DM, Baxter GF. Protecting the ischemic and reperfused myocardium in acute myocardial infarction: distant dream or near reality? Heart,2000,83:381-387.
[6] Kloner RA,Arimie RB,Kay GL,et al.Evidence for stunned myocardium in humans: a 2001 update.Coron Artery Dis,2001,12:349-356.
[7] Bolli R.The late phase of preconditioning.Circ Res,2000,87:972-983.
[8] Zhao ZQ, Corvera JS, Halkos ME,et al.Inhibition of myocardial injury by ischemic postconditioning during reperfusion:comparision with ischemic preconditioning. Am J Physio1,2003,285:579-588.
[9] Zhu M, Feng JH, Lucchinetti E,et al.Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvagekinase path way.cardiovascular research,2006,72:152-162.
[10]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[11] Jenings RB, Sommers HM, Smyth GA,et al.Myocardial necrosis induced by tempornary ocllusion of a coronary artery in the dog.Arch Pathol,1960,70:68-78.
[12]徐清斌,张馥敏,杨志健,等.局部注射自体骨髓间充质干细胞修复心梗后心肌的实验研究.江苏医药杂志,2004,30:266-267.
[13] Fung K,Weisel RD. Enhanced myocardial angiogenesis by gene transfer with transplanted cells.Circulation,2001,104:1218-1222.
[14]陈运贞.慢性心肌梗塞大鼠实验模型.重庆医科大学学报,2002,27:153-155.
[15]徐美清,何华等.犬急性心肌梗死模型制备的方法学研究.安徽医学,2002,23:6-8.
[16]杨国勋,刘唐威,钟国强,等.心肌梗死诊断中的应用.广西医学,2007,29:1135-1138.
[17] Tsang XL,Sato H,Tiwari S,et al.Cardioprotection by postconditioning in conscious rats is limited to coronary occlusions <45 min. Am J Physiol Heart Circ Physiol,2006, 291:H2308-H2317.
[18] Daring CE,Jiang R,Marnard M,et al.postcondition via stuttering reperfusion limits myocardial infact size in rabbit hearts role of ERK1/2.Am J Physol Heart Circ Physiol,2005,289:H1618-1626.
[19] David GD.Myocardial reperfusion injury:a new review.Cardiaovasc Res, 2004, 61:363-364.
[20] Galinnanes M, Fowler AG.Role of clinical pathologies in myocardial injury following ischemia and reperfusion. Cardiaovasc Res,2004,61:512- 521.
[21] Kato R,Foex P.Myocardial protection by anesthetic agents against ischemia-reperfusion injury:an update for anesthesiologists.Can J Anaesth, 2002,49:777-791.
[22] Rong F,Peng Z,Ye MX,et al.Myocardial apoptosis and infarction after ischemia/reperfusion are attenuated by kappa-opioid receptor agonist. Arch Med Res,2009, 40:227-234.
[23] Piao CS, Gao S, Lee GH, et al. Sulforaphane protects ischemic injury of hearts through antioxidant pathway and mitochondrial K(ATP) channels. Pharmacol Res, 2010,61:342-348.
[24] Gaugron P, Eilles C, Kugler L, et al. Progressive left ventricular dysfunction and remodeling after myocardial infarction potential mechanisms and early predictiors.Circulation,1993,87:755-763.
[25] Bolognese L,Ducci K,Angioli P,et al. Elevations in troponin I after percutaneous coronary interventions are associated with abnormal tissue level perfusion in high risk patients with non ST segment elevation acute oronary syndromes. Circulation,2004, 110:1592-1597.
[26] Zaugg M,Lucchinetti E,Garcia C,et al.Anaesthetics and cardiac precondition. Clinical implications.Br J Anaesth,2003,91:566-576.
[27] Philipp S, Downey M, Cohen MV,et al.Postconditioning must be initiated in less than 1 min following reperfusion and is dependent on adenosine receptors and PI3-kinase. Circulation,2004,110-l68.
[28] Ohtake M,Morino S,Kaidoh T,et,al.Three dimensional structural changes in cerebral microveasels after transient focal cerebral ischemia in rats scanning electron micoscopic study of corrosion casts.Neuropa theology,2004,24:219-227.
[29] Zatta AJ,Kin H,Lee G,et,al.Infarct sparing effect of myocardial postconditioning is dependent on protein kinase C signalling.Cardiovasc Res,2006,70:315-324.
[30] Li CM, Zhang XH, Ma XJ, et al. hearts with acute myocardial Impact of remote postconditioning on rabbit ischemic reperfusion injury.Chin J of Patho physiology,2006, 22:2332-2335.
[1] Milerova M,Charvatova Z,Skarka L,et al.Neonatal cardiac mitochondria and ischemia/reperfusion injury. Mol Cell Biochem. 2010,335:147-153.
[2] Honda HM,Korge P,Weiss JN. Mitochondria and ischemia reperfusion injury.Ann N Y Acad Sci,2005,1047:248-258.
[3] Maulik N,Yoshida T,Engelman RM,et al.Ischemic-preconditioning attenuates apoptotic cell death as sociated with ischemia/reperfusion.Mol Cell Biochem 1998,186:139-145
[4] Zhao ZQ, Corvera JS, Halkos ME, et al. Inhibition of myocardial injury by ischemic postconditioning during reperfusion: comparison with ischemic preconditioning. Am J Physiol Heart Circ Physiol,2003,285:H579-H588.
[5] Zhu M, Feng JH, Lucchinetti E,et al.Ischemic postconditioning protects remodeled myocardium via the PI3KPKB/ Akt reperfusion injury salvagekinase path way.cardiovascular research,2006,72:152-162.
[6] Schulz R,Boengler K,Dodoni G,Ruiz-Meana M,et al.Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.Cardiovasc Res,2005,67:234-244.
[7]曾玉杰,冯义柏,于世龙,等.卡维地洛和庚醇对心肌缺血再灌注损伤的保护作用.中华老年多器官疾病杂志,2007,6:123-127.
[8] Gavrieli Y,Sherman Y,Ben-Sasson SA.Identification of programmed cell death in situ via specific labelling of nuclear DNA fragmentation.J cell Biol, 1992,119:493-501.
[9] Flameng W, Borgers M, Daenen W,et al. Ultrastructural and cytochemical correlates of myocardial protection by cardiac hypothermia in man. J Thorac Cardiovasc Surg,1980,79:413-424.
[10] Emaus RK,Grunwald R,Lemaslers JJ.Rhodamine 123 as a probe of Iransmem brane potential in isolated rat liver mitochondria:spectra1 and metabolic properties.Biochim Biophys Acta,1986,850:436-448.
[11] Elizabeth AF,James DM,Takttro T,et al.Myocardial mitochondrial calcium accumulation modulates nuclear calcium accumulation and DNA Fragmentation.Amm Thorac Surg,1995,60:338-334.
[12]司徒镇强,吴军正,主编.细胞培养.第1版.西安:世界图书出版西公司,1996. 213-214.
[13] Machado NG, Alves MG, Carvalho RA, et al. Mitochondrial involvement in cardiac apoptosis during ischemia and reperfusion: can we close the box? Cardiovasc Toxicol,2009,9:211-227.
[14] Fliss H. Accelerated apoptosis in friend of foe. Basic Res Cardiol reperfusion myocardium,1998,93:90-96
[15] Zhang LP, Ma HJ, Bu HM, et al. Polydatin attenuates ischemia/reperfusion-induced apoptosis in myocardium of the rat.Sheng Li Xue Bao,2009,61:367-372.
[16] Castedo E,Segovia J,Escudero C,et al.Ischemia-reperfusion injury during experimental heart transplantation. Rev Esp Csrdiol,2005,58: 941-950.
[17] Gross GJ,Kersten JR,Warltier DC.Mechanisms of postischemic contractile dysfunction.Thorac Surg,1999,68:1898-904.
[18] Baumgartner HK,Gerasimenko JV,Thorne C,et al. Calcium elevation in mitochondria is the main Ca2+ requirement for mPTP opening. J Biol Chem, 2009,284:20796-20803.
[19] Novgorodov SA, Gudz TI.Ceramide and mitochondria in ischemia/reperfusion. J Cardiovasc Pharmacol,2009,53:198-208.
[20] Mattson MP,Kroeme G.Mitochondria in cell death:novel targets for neuroprotectionand cardioprotection.Trends Mol Med,2003,9:196-205.
[21] Arrell DK,Elliott ST, Kane LA,et al. Proteomic analysis of preconditioning: novel protein targets converge to mitochondria metabolism pathways.Circ Res,2006,99:706-714.
[22]吴青,陶红凯等.缺血再灌注诱导心肌细胞凋亡及凋亡相关基因表达的研究.中国心血管病研究杂志,2004,2:905-908.
[23]徐少东,马礼坤,屈朝法.犬急性心肌梗死晚期再灌注对心肌Fas/FasL系统的影响及其可能的氧化应激机制.中国病理生理杂志2007,23:307-310.
[24] Ratha J, Majumdar KN, Dhara K, et al. Attenuated Leishmanial sphingolipid induces apoptosis in A375 human melanoma cell via both caspase dependent and independent pathways. Mol Cell Biochem, 2007,304:143-154.
[25] Zanna C,Ghelli A,Porcelli AM,et al.Caspase independent death of Leber’s hereditary optic neuropathy cybrids is driven by energe tic failure and mediated by AIF and endonuclease G.Apoptosis,2005,10:997-1007.
[26] Li XZ,Liu JX.Role of autophagy in cardiac myocyte during ischemia/reperfusion.Chinese Pharmacological Bulletin,2008,24:704-707.
[27]杨兴易,石正蒙,顾桂国等.参附注射液对心肺复苏后大鼠心肌细胞保护作用的研究.临床急诊杂志,2005,6:16-19.
[28] Paradies G,Petrosillo G,Pistolese M,et a1.Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart:involvement of reactive oxygen species and cardiolipin.Circ Res,2004,94:53-59.
[1] Schulz R, Heusch G1.Connexin43 and ischemic preconditioning.Adv Cardiol,2006,42:213-227.
[2] Solan JL, Lampe PD. Connexin43 phosphorylation: structural changes and biological effects. Biochem J,2009419:261-272.
[3]辛莉,郭国庆,沈伟哉.间隙连接蛋白家族的研究进展.中国病理生理杂志, 2007,23: 1240-1243.
[4] Boengler K,Dodoni G,Rodriguez-Sinovas A,et al.Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic precondi tioning.Cardiovasc Res,2005,67:234-244.
[5] Goubaeva F, Mikami M,Giardina S,et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun,2007,352:97-103.
[6] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006,99:93-101.
[7] Saltman AE,Aksehirli TO,Valiunas V,et al.Gap junction uncoupling protects the heart against ischemia.J Thorac Cardiovase Surg,2002,124:371-376.
[8]陈宝平,夏强,沈岳良.缝隙连接失耦剂Heptanol对缺血心肌的影响,中国心脏起搏与心电生理杂志,2002,16:139-142.
[9] Carcia- Dorado D, Theroux P, Desco M, et al. Cell-to-cell interaction:amechanism to explain wave-front progression of myocardial necrosis. Am physio soc, 1989,214: H1266-1273.
[10]苏德淳,常志文,范书英.缝隙连接在大鼠缺血预适应心肌保护中的作用.中华心血管病杂志,2006,34:690-694.
[11] Schwanke U,Konietzka I,Duschin A, et al. No ischemic preconditioning in heterozygous connexin 43 deficient mice.Am J Physiol Heart Cire Physiol, 2002,283:1740-1742.
[12] Daleau P, Boudriau S, Michaud M, et al. Preconditioning in the absence or presence of sustained ischemia modulates myocardial Cx43 protein levels and Pharmacol ,2001,79:371-378.
[13] Quist AP, Rhee SK, Lin H, et al. Physiological role of gap functional hemichannels extracellular calcium-dependent volume regulation. J Cell Biol,2000,148:1063-1074.
[14]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[15] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006, 99: 93–101.
[16] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol, 2007, 292: H1764-H1769.
[17] Schwanke U, Konietzka I, Duschin A, et al. No ischemic preconditioning in heterozygous connexin43-deficient mice. Am J Physiol Heart Circ Physiol,2002, 283: H1740–H1742.
[18] Cruciani V, Mikalsen SO. Connexins, gap junctional intercellular communication and kinases.Biol Cell,2002,94:433–443.
[19] Evans WH, Martin PE. Gap junctions:Structure and function.Mol Membr Biol, 2002,19:121-125.
[20] Jeyaraman M,Tanguy S,Fandrich RR,et al.Ischemia-induced dephosphorylation of cardiomyocyte connexin 43 is reduced by okadaie acid and calyculin A but not fostriecin.Mol Cell Biochem,2003,242:129-134.
[21] De MA,Vega VL,Contreras JE. Gap junctions,homeostasis,and injury. J Cell Physiol,2002,191:269–282.
[22] Goubaeva F,Mikami M,Giardina S,et al.Cardiac mitochondrial connexin 43 regulates apoptosis.Biochemical and Biophysical Research Communications,2007,352:97-103.
[23] Heinzel FR, Luo YK, Li XK, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice. Circ Res,2005,97:583-586.
[24] Boengler K, Konietzka I , Buechert A,et al. Loss of ischemic preconditioning cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43.Am J Physiol Heart Circ Physio1,2007,292:H1764-H1769.
[25] Boengler K,Dodoni G,Rodriguez-Sinovas A,et al.Connexin 43 in cardiomyocytemitochondria and its increase by ischemic precondi tioning.Cardiovasc Res,2005,67:234-244.
[26] Boengler K, Ruiz-Meana M, Miro-Casas B, et al.Connexin 43 controls mitochondrial respiration. Circulation ,2006, 114:243-251.
[27] Miro-Casas R, Ruiz-Meana M, Agullo R, et al. Connexin43 in the inner mitochondrial membrane of cardiomyocytes forms hemichannels that contribute to matrix volume regulation. Circulation,2007,116-167.
[28] Saffitz JE,Laing JG,Yamada KA,et a1.Connexin expression and turnover :implications for cardiac excitability.Circ Res,2000,86:723-728.
[29] Fernandez-Cobo M,Gingalewski C,Dmjan D.Down regrudation of connexin 43 gene expression in rat heart during inflammation:The role of tumor necrosis factor.Cytokine,l999,ll:216-224.
[30] Solan JL,Lampe PD.Connexin43 phosphorylation: structural changes and biological effects.Biochem J,2009,419:261-272.
[1] Naitoh K, Ichikawa Y, Miura T,et al.MitoKATP channel activation suppresses gap junction permeability in the ischemic myocardium by an ERK-dependent mechanism. Cardiovasc Res,2006,70:374-83.
[2] Mykytenko J, Reeves JG, Kin H, et al.Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K(ATP) channels during reperfusion. Basic Res Cardiol,2008,103:472-484.
[3] O'Rourke B.Evidence for mitochondrial K+ channels and their role in cardioprotection.Circ Res,2004,94:420-432.
[4] Kamada N,Kanaya N,Hirata N,et al. Cardioprotective effects of propofol in isolated ischemia-reperfused guinea pig hearts: role of KATP channels and GSK-3beta. Can J Anaesth,2008,55:595-605.
[5] Simerabet M, Robin E, Aristi I, et al.Preconditioning by an in situ administration of hydrogen peroxide: involvement of reactive oxygen species and mitochondrial ATP-dependent potassium channel in a cerebral ischemia-reperfusion model. Brain Res,2008,1240:177-184.
[6] Korge P,Honda HM,Weiss JN.K+ -dependent regulation of matrix volume improves mitachondrial function under conditions mimicking ischemia reperfusion.Am J Physiol Heart Circ Physiol,2005,289:H66-H77.
[7] Yang XM,Proctor JB,Cui L,et al.Multiple,brief coronary occlusions during early reperfusion protect rabbit hearts by targeting cell signaling pathways . Am Coil Cardiol,2004,44:l103-1110.
[8] Dhein S. Gap ,junction channels in the cardiovascular system: pharmacological And physiological modulation.TrendsPharmacol Sci,1998,19:229-241.
[9] Kevelaitis P,Peynet J,Mouas C,et al.Opening of potassium channels:The common cardioprotective link between preconditioning and natural hibernation? Circulation,1999,99:3079-3085.
[10] Quist AP,Rhee SK,Lin H,et al.Physiological role of gap ,functional Hemichannels.Bxtracellular calcium-dependentisosmotic volume regulation.JCell Biol,2000,148:1063-1074.
[11] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 tothe inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res, 2006, 99: 93–101.
[12] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection. Circ Res,2006,99:93-101.
[13] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43. Am J Physiol Heart Circ Physiol, 2007, 292: H1764-H1769.
[14] Heinzel FR, Luo Y, Li X, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res,2005,97:583-586.
[15] Penna C, Mancardi D,Raimondo S, et al. The paradigm of postconditioning to protect the heart.Cell Mol Med,2008,12: 435–458.
[16]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[17] Wojtovich AP, Burwell LS, Sherman TA, et al. The C. elegans mitochondrial K+(ATP) channel: a potential target for preconditioning. Biochem Biophys Res Commun,2008,376:625-628.
[18] Mykytenko J,Reeves JG,Kin H,et al.Persistent beneficial effect of postconditioning against infarct size: role of mitochondrial K(ATP) channels during reperfusion.Basic Res Cardiol,2008,103:472-484.
[19] Krolikowski JG, Bienengraeber M,Weihrauch D,et al. Inhibition of mitochondrial permeability transition enhances isoflurane-induced cardioprotection during early reperfusion:the role of mitochondrial KATP channels.Anesth Analg,2005,101: 1590-1596.
[20] Quinlan CL, Costa AD, Costa CL, et al. Conditioning the heart induces formation of signalosomes that interact with mitochondria to open mitoKATP channels. Am J Physiol Heart Circ Physiol,2008,295:H953-H961.
[1]辛莉,郭国庆,沈伟哉.间隙连接蛋白家族的研究进展.中国病理生理杂志,2007: 1240-1243.
[2] Schulz R, Heusch G.Connexin 43 and ischemic preconditioning.Cardiovasc Res, 2004, 62: 335–344.
[3] Rodriguez-Sinovas A, Boengler K, Cabestrero A, et al. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.Circ Res,2006,99: 93–101.
[4] Boengler K, Dodoni G, Rodriguez-Sinovas A, et al. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning.CardiovascRes,2005,67:234–244.
[5] Ruiz-Meana M, Rodríguez-Sinovas A, Cabestrero A,et al.Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischemic reperfusion injury.Cardio vascular Research,2008,77:325-333.
[6] Palmer JW, Tandler B, Hoppel C, et al. Biochemical properties of subsarcolemmal and inter-fibrillar mitochondria isolated from rat cardiac-muscle. J Biol Chem,1977,252: 8731–8739.
[7] Boengler K, Stahlhofen S, van de Sand A, et al. Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria. Basic Res Cardiol, 2009, 104: 141-147.
[8] Li H, Brodsky S, Kumari S, et al. Paradoxical overexpression and translocation of connexin 43 in homocysteine-treated endothelial cells. Am J Physiol Heart Circ Physiol, 2002, 282: H2124–H2133.
[9] Schulz R, Boengler K, Totzeck A, et al. Connexin 43 in ischemic pre- and postconditioning. Heart Fail Rev, 2007, 12: 261–266.
[10] Totzeck A, Boengler K, van de Sand A, et al. No impact of protein phosphatases on connexin 43 phosphorylation in ischemic preconditioning[J]. Am J Physiol Heart Circ Physiol, 2008, 295(5): H2106-2012.
[11] Boengler K, Konietzka I, Buechert A, et al. Loss of ischemic preconditioning's cardioprotection in aged mouse hearts is associated with reduced gap junctional and mitochondrial levels of connexin 43.Am J Physiol Heart Circ Physiol,2007,292: H1764-H1769.
[12] Schulz R, Gres P, Skyschally A,et al. Ischemic preconditioning preserves connexin 43 phosphorylation during sustained ischemia in pig hearts in vivo. FASEB J,2003,17: 1355–1357.
[13] Dupont E, Matsushita T, Kaba RA, et al. Altered connexin expression in human congestive heart failure. J Mol Cell Cardiol, 2001, 33: 359–371.
[14] Doble BW, Dang X, Ping P, et al. Phosphorylation of serine 262 in the gap junction protein connexin-43 regulates DNA synthesis in cell– cell contact forming cardiomyocytes. J Cell Sci, 2004,117: 507– 514.
[15]罗红鹤,雷艺炎,陈振光,等.缺血预处理在心瓣膜置换术中对心电活动及细胞缝隙连接的保护作用.中国病理生理杂志,2008,24:1953-1956.
[16] Schwanke U, Konietzka I, Duschin A, et al. No ischemic preconditioning in heterozygous connexin43-deficient mice.Am J Physiol Heart Circ Physiol,2002,283: H1740–H1742.
[17] Heinzel FR, Luo Y, Li X, et al. Impairment of diazoxide-induced formation of reactive oxygen species and loss of cardioprotection in connexin 43 deficient mice. Circ Res, 2005, 97: 583–586.
[18] Gutierrez PL. The role of NAD(P)H oxireductase (DT-diaphorase) in the bioactivation of quinone-containing antitumor agents: a review.Free Radic Biol Med, 2005, 29: 263–275.
[19] Heusch G, Büchert A, Feldhaus S, et al. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol, 2006, 101:354–356.
[20] Penna C, Mancardi D,Raimondo S, et al. The paradigm of postconditioning to protect the heart . Cell Mol Med, 2008, 12: 435–458.
[21]何燕,曾志羽,钟国强,等.线粒体Cx43参与缺血后处理对兔急性心肌缺血再灌注损伤保护作用.中华心血管病杂志, 2010,38:357-362.
[22]陈宝平,毛红娇,范芳燕,等.缝隙连接脱偶联剂庚醇对大鼠缺血-再灌注损伤心肌的保护作用.中国病理生理杂志,2005,21:1071-1075.
[23]何燕,曾志羽,钟国强,等.线粒体Cx43参与Heptanol预处理对兔急性心肌缺血再灌注损伤保护作用.中国药理学通报, 2009,25:1660-1665.
[24] Miro-Casas E, Ruiz-Meana M, Agullo E, et al. Connexin43 in cardiomyocyte mitochondria contributes to mitochondrial potassium uptake.Cardiovasc Res,2009,83: 747-756.
[25] Goubaeva F, Mikami M, Giardina S, et al. Cardiac mitochondrial connexin 43 regulates apoptosis. Biochem Biophys Res Commun, 2007, 352: 97-103.