Role Of Mitochondria And Mitochondrial Cx43 On The Myocardial Ischemia/Reperfusion Injury After Ischemic Postconditioning And Heptanol Preconditioning In Rabbit

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 rabbitsObjective: 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 rabbitObjective: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 injuryObjective: To investigate the effects of ischemic postconditioning and Heptanol preconditioning on Cx43 of membrane and mitochondria in rabbit myocardial ischemia-reperfusion injuryMethods: 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 4Impact of PC and heptanol pretreatment involved in mitochondrial ATP-sensitive potassium channel on Cx43 of mitochondria in rabbit ischemia-reperfusion injuryObjective: 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.