Research On The Effect Of LDHA On Myocardial Apoptosis In The Ischemia Reperfusion Injury

With the popularity and implementation of emergency intervention or emergency thrombolytic therapy in recent years,a large number of patients with acute myocardial infarction(AMI)have not only saved their lives,but also improved their prognosis.However,the ischemia/reperfusion(I/R)injury is always a thorny issue in the reperfusion therapy of AMI.A large number of studies have confirmed that there is a clear myocardial injury after the reperfusion therapy which often cause cardiomyocyte apoptosis,left ventricular remodeling,heart failure and arrhythmia and so on.How to reduce myocardial ischemia/reperfusion injury,reduce apoptosis and necrosis become one of the hot spots in coronary heart disease research at present.Our research focused on the intervention of apoptosis after myocardial infarction to improve the survival rate of patients with AMI and improve prognosis.Lactate dehydrogenase A(LDHA)is an important enzyme in the glucose metabolism.It is involved in the process of glycolysis that is the conversion of pyruvate to lactic acid.However,it has been found that LDHA can participate in cell proliferation and apoptosis,especially in lung cancer,cholangiocarcinoma and other tumor diseases.It has been found that overexpression of LDHA can reduce apoptosis and promote tumor cell proliferation,which may be related to that tumor cells in the relative lack of energy during growth,increased LDHA expression of the tumor self-protection mechanism for cells to adapt to energy metabolism.Since LDHA is involved in cell proliferation and apoptosis,we hypothesized that LDHA may have a relationship with apoptosis after AMI in patients with coronary heart disease,and may be a target of an intervention of apoptosis after reperfusion therapy.This study first analyzed the phenomenon and the corresponding factors of apoptosis after reperfusion in AMI patients;By making the model of myocardial ischemia/reperfusion injury in rats,our study further confirmed the existence of definite apoptosis after reperfusion in AMI,accompanied by increased expression of cellular LDHA,presumably increased LDHA expression may be related to the adaptive protection of ischemia/reperfusion injury.Finally,we further made the hypoxia/reoxygenation(H/R)injury modeling of primary cardiomyocytes and deeply studied the effect of LDHA on myocardial apoptosis in the ischemia reperfusion injury.It can provide theoretical basis and new ideas for the prevention and clinical treatment of ischemia/reperfusion injury in patients with AMI.Part one Changes of Serum Apoptotic Proteins Caspase-3,Bax and Bcl-2 after PCI in Acute Myocardial Infarction Patients and Related FactorsObjective: To study the changes of serum caspase-3,Bax and Bcl-2 in patients with AMI after emergency PCI and to analyze the related factors in order to clarify the influencing factors of apoptosis after reperfusion in patients with AMI.Methods: The patients were screened strictly according to the standard and divided into AMI group and control group.Serum levels of caspase-3,Bax and Bcl-2 were detered by enzyme-linked immunosorbent assay(ELISA)on the third day after operation.Differences between the two groups were compared and factors affecting apoptotic proteins were analyzed according to subgroups.Minewhile,Differences in myocardial enzymes(CK-MB and cTnI)and left ventricular function(LVESVI,LVEDVI,LVEDD and LVEF)were compared between the two groups.Grouping:(1)Acute myocardial infarction group(AMI Group): The inclusion criteria were first diagnosed patients with AMI with symptoms of myocardial infarction over 30 min and persistent ST elevation(?1 mm)in two or more adjacent leads in the ST segment or new appeared left bundle branch block,while patients with subsequent myocardial enzyme confirmed increased.There were 53 cases of patients with 33 males and 20 females;21 cases were less than 60 years old,20 cases were 60-70 years old and 12 cases were ?70 years old,with an average age of(62±10)years;15 cases had diabetes at the same time,hypertension in 20 cases;perfusion time ? 6h in 25 cases,6-12 h in 28 cases;single branch lesion in 24 cases,double branch lesion in 29 cases;anteroseptal wall infarction in 13 cases,anterior infarction in 12 cases,inferior wall infarction in 15 cases of infarction and posterior wall infarction in 13 cases.(2)Control Group: Emergency coronary angiographies in 48 patients hospitalized with chest pain had been performed.The result was that coronary arteries had no obvious stenosis,or stenosis,but with TIMI grade flow 3.There were 29 males and 19 females cases among the 48 patients,mean age(60 ± 9)years,of which 12 cases were suffering from diabetes at the same time,with hypertension in 17 cases.Result:1.There was a significant difference in the contents of caspase-3 and Bax between the two groups(P<0.05),and the difference was statistically significant.The serum levels of caspase-3 and Bax in the patients of AMI Group were significantly higher.The Bcl-2 content was no difference between the two groups(P>0.05).The two groups of patients were significantly different in myocardial enzymes(CK-MB and cTnI)and left ventricular function(LVESVI,LVEDVI,LVEDD and LVEF)(P<0.05).Myocardial enzymes in the patients of AMI Group were significantly higher,meanwhile left ventricular function index decreased significantly compared with the control group.2.Analysis of subgroups in the AMI Group showed that the three apoptotic proteins levels had no difference in different age groups,gender groups,hypertension groups and diabetes groups(P>0.05).However,the contents of three apoptotic proteins in different reperfusion time groups were significantly different(P<0.05),and the difference was statistically significant.caspase-3 and Bax were significantly increased in reperfusion group 6-12 hours.The levels of Bcl-2 was obvious reduction.3.The subgroup analysis also suggested that the serum levels of apoptosis proteins in different infarction groups were different(P<0.05),the difference was statistically significant.SNK test showed that there is significantly difference in three apoptotic protein content between the anterior wall group and other every group(P<0.05),there was no significant difference between the other two groups(P>0.05),indicating that the serum levels of apoptotic proteins in patients with AMI were significantly different from those in other groups.Among them,caspase-3 and Bax was significantly increased,Bcl-2 was significantly lower.Conclusion:1.Myocardial infarction patients have definite myocardial necrosis and apoptosis,and associated with decreased cardiac function.2.When the reperfusion treatment time is different,the occurrence of myocardial cell apoptosis is different.The later the reperfusion time is,the more obvious the occurrence of apoptosis is.3.When extensive anterior myocardial infarction occurrs,myocardial apoptosis is more obvious.Part two Study on the Effect of Myocardial Ischemia/Reperfusion Injury on Myocardial Apoptosis and Expression of LDHA in RatsObjective: To study the effect of myocardial ischemia/reperfusion injury on myocardial apoptosis and expression of LDHA in rat model of myocardial ischemia/reperfusion injury.Methods: Male Wistar rats were used to establish the model of AMI ischemia/reperfusion by ligating and releasing the anterior descending artery to observe the expression of LDHA and the apoptosis in myocardial tissue.Grouping:(1)Control Group: The heart was exposed by thoracotomy,the anterior descending artery was found between the aorta and the left atrial appendage,and the ligament was not ligated at a distance of about 2-3 mm from the anterior descending artery.(2)I/R Group: The heart was exposed by thoracotomy.The anterior descending artery was found between the aorta and the left atrial appendage.The anterior descending artery blood flow was occluded for 30 min at the same position by cannula,and then reperfused for 120 min.Then the area of myocardial ischemic necrosis was observed by staining,the apoptosis index was calculated by TUNEL method,and the expressions of caspase-3,Bax,Bcl-2 and LDHA were detected by Western Blot.Real-time PCR was used to detect the mRNA expression level of LDHA.Result:1.From the infarct area,myocardial ischemic area and infarct area in I/R group were much larger than those in Control group(P<0.05),and there was significant difference between the two groups,which was statistically significant.TUNEL staining also showed that compared with Control group,Apoptosis index in I/R group was significantly increased(P<0.05),the difference was statistically significant.2.Western Blot results showed that compared with Control group,the expression of caspase-3 and Bax in I/R group were significantly increased(P <0.05),while the expression of Bcl-2 protein in I/R group was significantly decreased(P<0.05),The differences were statistically significant.Thus,the expression of proapoptotic proteins caspase-3 and Bax in rat myocardial cells in myocardial ischemia/reperfusion injury increased,and the expression of anti-apoptotic protein Bcl-2 decreased,thereby promoting apoptosis.3.Western Blot results also showed that compared with Control group,the expression level of LDHA in I/R group was significantly increased(P<0.05),the differences was statistically significant.4.The results of PCR showed that compared with Control group,the expression level of LDHA mRNA in I/R group was significantly increased(P <0.05),and the difference was statistically significant.In conclusion,the gene expression of LDHA mRNA in rat myocardial cells in myocardial ischemia/reperfusion injury increased,and the production of LDHA increased,which may be closely related to apoptosis.Conclusion:1.Myocardial ischemia/reperfusion injury showed obvious myocardial necrosis and apoptosis in rats.2.The expression of apoptosis proteins caspase-3 and Bax in myocardial tissue of rats in myocardial ischemia/reperfusion injury increased,and the expression of anti-apoptotic protein Bcl-2 decreased,thereby promoting apoptosis.3.The increase of expression of LDHA and LDHAmRNA in myocardial tissue of rats in myocardial ischemia/reperfusion injury may be closely related to apoptosis.Part three Effects of target regulation of LDHA through the PDK1/Akt/mTOR pathway on myocardial apoptosis caused by ischemia/ reperfusion injury in ratsObjective: To investigate the effects of LDHA on cardiomyocyte apoptosis in myocardial ischemia/reperfusion injury and to investigate the molecular mechanism of LDHA's effect on ischemia/reperfusion cardiomyocyte apoptosis in rats through PDK1 / Akt / mTOR pathway.Methods: Primary myocardial cells of neonatal Wistar rats were used to simulate myocardial ischemia/reperfusion injury by hypoxia/reoxygenation injury modeling of primary cardiomyocytes.The rats were subjected to 3 h hypoxia and 3 h reoxygenation.Grouping:(1)Blank control group + no vector group(control group): Cardiomyocytes were cultivated under normoxic condition and transferred with pcDNA3.1(+)vector by lipo2000.(2)Blank control + LDHA transfection group(Control + LDHA group): Cardiomyocytes were cultivated under normoxic condition,and transferred with pcDNA3.1(+)-LDHA vector by lipo2000.(3)Model + no vector group(H/R group):After being transfected of pcDNA3.1(+)vector,the primary cardiomyocytes were performed reoxygenation for 3 h after 3h of hypoxia.(4)Model + LDHA transfection group(H/R+LDHA group): After being transfected of pcDNA3.1(+)-LDHA,the primary cardiomyocytes were performed reoxygenation for 3 h after 3h of hypoxia.Cardiomyocyte proliferation was detected by microplate reader.Cardiomyocyte apoptosis was detected by flow cytometry.Protein expression was detected by Western blot.Real-time PCR was used to detect mRNA expression.Dual-luciferase assay was used to detect the target gene.Result:1.After primary cardiomyocytes were cultivated for 72 h,the OD value of primary cardiomyocytes detected by microplate reader in H/R group was(0.84±0.08),which was significantly lower than that in Control group(1.23±0.11,P<0.05).The difference was statistically significant.After primary cardiomyocytes were cultivated for 72 h,the apoptosis rate of primary cardiomyocytes detected by flow cytometry in H/R group was(44.63 + 7.25)%,which was significantly higher than that in Control group(7.12±1.82)%,There was a significant difference between groups(P <0.05).2.After primary cardiomyocytes were cultivated for 72 h,the expression of LDHA in H/R group was significantly higher than that in Control group by Real-time PCR and Western blot.The relative expression of LDHA mRNA and protein in H/R group were(9.879±2.021)and(0.988 ± 0.114)respectively.The relative expression levels of LDHA mRNA and protein in the control group were(2.289±1.261)and(0.287±0.165),respectively,and the differences were statistically significant(P<0.05).3.Cardiomyocytes were cultivated for 72 h.Western blot results showed that AKt(1.098± 0.274),mTOR(1.124±0.332)and PDK1(0.934 ±0.246)in H/R group were significantly increased compared with Control group,The difference was statistically significant(P<0.05).4.The results of microplate reader showed that the OD value in the H/R + LDHA group(1.140 ±0.075)was significantly higher than that in the H/R group(0.84±0.08),the difference was statistically significant(P=0.000),indicating that LDHA overexpression significantly promoted cardiomyocyte proliferation.The results of flow cytometry showed that the rate of cardiomyocyte apoptosis was(26.27+4.35)% in H/R+LDHA group and(44.63 + 7.25)% in H/R group.The difference was statistically significant(P=0.000),indicating that LDHA over-expression significantly inhibited cardiomyocyte apoptosis.5.Under the conditions of H/R,the cardiomyocytes transfected with LDHA over-expression vector were cultivated for 72 h.The results of Western blot showed that the protein expressions of AKT(3.454±0.782),mTOR(3.793±1.122)and PDK1(2.814±0.948)were significantly increased in H/R+LDHA group compared with those in H/R group.the differences were statistically significant(P = 0.000).6)Dual luciferase results showed that the luciferase signal intensity of vectors carrying wild type PDK1 reporter gene decreased by 45% compared with the control group after the transfection with LDHA overexpression vectors,while the luciferase signal intensity of the vectors carrying mutant type PDK1 reporter gene showed no significant change,indicating that the PDK1 gene is the target of LDHA.Conclusion:1.After primary cardiomyocytes were cultivated for 72 h,primary cardiomyocytes apoptosis in H/R group increased significantly.2.After primary cardiomyocytes were cultivated for 72 h,the expression of LDHA and LDHA mRNA in primary cardiomyocytes of H/R group increased,which may be the self-protection of apoptosis.3.After primary cardiomyocytes were cultivated for 72 h,Akt,mTOR and PDK1 protein expression of primary cardiomyocytes in H/R group increased significantly,may be self-protection mechanism of apoptosis caused by ischemia/reperfusion injury.4.Overex-pression of LDHA can significantly increase the expression of AkT,mTOR and PDK1 in cardiomyocytes,promote the proliferation of cardiomyocytes and decrease the apoptosis.It indicates that LDHA overexpression inhibits cardiomyocyte apoptosis through AkT,mTOR and PDK1 protein.5.Dual luciferase method confirmed that PDK1 gene is the target of LDHA.Conclusion:1.Myocardial infarction patients have definite myocardial necrosis and apoptosis,and associated with decreased cardiac function.The later the reperfusion time is,the more obvious the occurrence of apoptosis is.When extensive anterior myocardial infarction occurrs,myocardial apoptosis is more obvious.2.Myocardial ischemia/reperfusion injury showed obvious myocardial necrosis and apoptosis.The expression of apoptosis proteins caspase-3 and Bax in myocardial ischemia/reperfusion injury increased,and while the expression of anti-apoptotic protein Bcl-2 decreased.3.The increase of expression of LDHA and LDHAmRNA in myocardial ischemia/reperfusion injury may be closely related to apoptosis.4 The hypoxia/reoxygenation injury modeling of primary cardiomyocytes could increase primary cardiomyocytes apoptosis and Akt,mTOR and PDK1 protein expression of primary cardiomyocytes,accompanied by the expression of LDHA and LDHA mRNA of primary cardiomyocytes.5.Overexpression of LDHA can significantly increase the expression of AKT,mTOR and PDK1 in cardiomyocytes,inhabit cardiomyocytes apoptosis.It was confirmed that PDK1 gene is the target of LDHA.