| [Background and objective]Acute Myocardial Infarction (AMI) has become an important cause of increased mortality rates of cardiovascular disease. Malignant Arrhythmia and heart failure after Myocardial infarction is a common cause of death in patients with Myocardial infarction. Myocardial Ischemic Preconditioning (IP) is the ability to reduce the long-term harmful Ischemia Reperfusionon Myocardial damage after repeatedly transient Ischemia and Reperfusion. Studies have shown that IP can suppress the ventricular remodeling, reduce the incidence of arrhythmia and reduce the area of Myocardial infarction, but the mechanism is not completely clear yet. It has also been reported that Cardio-protection after MI with IP is related to the enhanced expression of Angiogenic growth factors, such as Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factors (bFGF). IP-induced protection also depends on functional Connexin 43 (Cx43)-formed channels. Cx43 plays an essential role in IP of the heart, since Cx43 deficiency decreases Myocardial tolerance to Ischemia-Reperfusion injury and abolishes infarct size reduction induced by IP after MI. However, the exact underlying intrinsic relationship between Cx43-mediated protection, anti-inflammation and Angiogenesis on Antiarr-Hythmic effects induced by IP remains to be established. Researches have shown that, Ischemic preconditioning can trigger the protective mechanism of the body, such as Adenosine, Bradykinin and Endothelin increases, which activate the Reperfusion injury salvage Kinase pathway through the corresponding receptors, PI3K/Akt pathway, leading to increased gene transcription and expression of effect. This increase plays the role in anti-Ischemia Reperfusion injury. Our previous experiments showed that the Angiogenesis after Hypoxia micro environment, the formation of collateral circulation of these effects is related to the PI3K/Akt pathway. Although the PI3K pathway in Ischemic preconditioning in the heart protective effect is very important, the PI3K/Akt signal Transduction pathway in the mechanism of Myocardial infarction on the Ischemic preconditioning Antiarrhythmic effect is not very clear yet.We research on the involvement of Phosphat Idylinositol 3-Kinase (PI3K)/Akt in the IP-like effects of Cx43 and Proangiogenic factors, specially focus on the IP role in protecting from Ischemia-induced arrhythmia, heart failure, and Myocardial remodeling. Herein, we investigate different Ischemia models, the protective effect of IP against ventricular arrhythmia and sudden cardiac death and the changes in Akt signaling to determine if Cx43 and Proangiogenic factors mediate these effects. The main aim of this work is to explore the possible mechanism of Ischemic preconditioning in anti arrhythmia and improvement malignant ventricular cardiac function. Then we provide new prospect for the prevention and therapy of Arrhythmia and heart failure after Myocardial infarction.[Methods]Experiment grouping methods:In the inducted Myocardial Infarction (MI) model,10 pigs from the 90 experimental pigs are selected randomly. These 10 pigs are open chest operated and Sham Ligation of LAD coronary artery, as control group. The other 80 pigs are randomized to receive local intramuscular injection of Phosphate Buffer Saline group (PBS) or local injection of PI3K inhibitor LY294002 group. Among the 80 pigs,20 pigs are randomly assigned to receive IP or no IP group. The forthcoming experimental animal are randomly divided into five groups:control group (n= 10), LI (anterior descending coronary Artery Ligation) group (N= 20), IPLI (Ischemic preconditioning and ligation of LAD) group group(N= 20), LYLI (injection of LY294002 and Ligation of LAD) group (N= 20) and LYIPLI (injection of LY294002 plus ischemic preconditioning and ligation of LAD) group (n= 20).Research methods:In the experiment, the experimental pigs undergo Echo-cardiographic examination, the LVEF, LVFS, LVEDIVST, LVEDPW, LVEDV, LVESV, LVEDD and LVESD values are measured.Beforehand, the induction of MI model,10 randomly selected normal experimental pigs are open chest operation and sham ligation of LAD coronary artery, which are taken as control group.80 other experimental pigs are randomized to receive local intramuscular injection of PBS or local injection of PI3K inhibitor LY294002 groups,20 experimental pigs, which were randomly assigned to receive IP or no IP group. Next, all the animal by ligation of left anterior descending branch of coronary artery LAD induced in the MI model, Intraoperative and postoperative recording pig Arrhythmia, including the starting time and the cumulative time.60 minutes after operation, conduct" the ECG, Echocardiographic, Coronary angiography (CAG) to confirm the Vascular Occlusion. Thirdly, keeping record for 7 days, after Echocardiographic examination measured LVEF, LVFS, LVEDIVST, LVEDPW, LVEDV, LVESV, LVEDD, and LVESD values. Ultimately, kill pigs and remove the Myocardial. Using staining methods such as H&E staining, chlorinated the three Phenyl four Triazole staining (TTC staining), and Masson trichrome staining (Masson’s trichrome stain), to investigate the IP on Myocardial tissue remodeling after Myocardial Infarction, inflammation, infarct size, influence and Collagen content. Using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) to detect quantitatively mRNA levels of Akt and Cx43 within 7 days after Myocardial Infarction. Employing the Western Blot Electrophoretic to display each level of Akt protein, phosphorylation of Akt Ser473, pAkt Thr308 (PAKT) and the horizontal of Cx 43. Then calculating the ratio of pAkt Thr308 versus pAkt Ser473 with total Akt. Employing the Immuno-Histo-Chemistry to observe the expression of Akt and Cx43 in the in local and infarct area. Meanwhile, employing the RT-PCR to analyze quantitatively of the Ang-1, bFGF and VEGF mRNA expression level. The expression level of Ang-1, bFGF and VEGF protein with7 days after ligation of LAD are detected in Western blot. The Immuno-Histo-Chemical staining shows the local expression of VEGF, Ang-1, bFGF after Myocardial Infarction 7 days. Finally, analyzing the peripheral vascular density of each infarction after Myocardial infarction employing vascular factor VIII antibody staining.[Results]1. Ischemic Preconditioning can significantly reduce the occurrence of animal mortality and malignant ventricular Arrhythmia 7 days after Myocardial Infarction, extending the first malignant ventricular Arrhythmia after Myocardial Infarction attack interval, malignant ventricular arrhythmia cumulative attack time. Application of PI3K inhibitor LY294002 abolish these therapeutic effect.2. Ischemic Preconditioning significantly increases LVEF and LVFS after Myocardial infarction, and improve left ventricular remodeling index (LVEF, LVFS, LVEDIVST, LVEDPW, LVEDV, LVESV, LVEDD and LVESD). The use of PI3K inhibitor LY294002 significantly decreases LVEF, LVFS value, the promotion of left ventricular remodeling.3. Ischemic Preconditioning reduces Myocardial Inflammation, infarct size, and collagen area. Increased infarct area of surviving Myocardium survival number. The use of PI3K inhibitor LY294002 of the infarct Myocardial remodeling deterioration, manifested as increased inflammation, collagen content and Myocardial Infarction area, reducing the infarction area of Myocardial viability.4. IP promotion of Akt expression level increases after myocardial infarction, promotes the Phosphorylation of Akt, up-regulates Cx43 expression in Myocardial tissue, improves myocardial structural remodeling. Application of PI3K inhibitor LY294002 significantly inhibits the expression of Akt, decreases Akt phosphorylation and the expression of Cx43 expression, inhibit Myocardial structural remodeling.5. IP promotes the expression of vascular infarction Myocardium growth factors of Ang-1, bFGF and VEGFmRNA and protein levels, and the use of PI3K inhibitor LY294002 reduces expression of these factors.6. IP promotes angiogenesis in the infarcted heart and formation of collateral circulation, and the use of PI3K inhibitor LY294002 attenuates this effect.[Conclusions]These findings demonstrate the Cardioprotective effects of IP on anti-ventricular arrhythmia and Myocardial repair through up-regulation of Akt-mediated Cx43 and growth factor signaling. |
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