Mechanism Research Of Targeting Activation Of ERK1/2 Signaling Pathway In The Prevention And Treatment Of Atherosclerotic Myocardial Ischemia/Reperfusion Injury

Objective:The aim of this study was to investigate the mechanisms and effects of targeting the extracellular signal-regulated kinase ERK1/2 signaling pathway in arteriosclerosis（AS）myocardial inducing ischemia/reperfusion（I/R）injury.To explore whether exogenous targeted transduction of constitutively active MEK1（CaMEK）gene can inhibit I/R injury by activating ERK1/2 pathway,regulating autophagy level,inhibiting cardiomyocyte apoptosis,and improving mitochondrial function.The main study included:（1）ApoE gene knockout(ApoE^-/-)mice were formed into a stable atherosclerosis model by a high-fat diet.To compare and study the difference of myocardial protective effects of ischemic post-conditioning（IPost）on healthy mice and AS mice,and its effects on the activity of ERK1/2 signaling pathway.（2）Recombinant double strand adeno-associated virus serotype 9 carrying CaMEK（dsAA9-CaMEK）virus was transfected in vitro,and the hypoxia/reoxygenation（H/R）model and oxidative stress model of neonatal rat myocardial cells were used to investigate the role and molecular mechanism of CaMEK overexpression in alleviating H/R injury and oxidative stress injury of myocardial cells.（3）Targeting transduction of CaMEK gene into myocardium of AS mice,through the in vitro and in vivo myocardial I/R model,explored the role of CaMEK gene overexpression in alleviating AS myocardial I/R injury and its molecular mechanism.Methods:（1）Eight weeks of C57BL/6J and ApoE^-/-male mice were selected as subjects.The mice were fed with conventional diet and high-fat diet for 4 months to detect the degree of aortic atherosclerosis in ApoE^-/-mice.In vivo I/R models and IPost models were established for both mice.The indicators were myocardial infarct size,myocardial enzymes,cardiomyocyte apoptosis,and expression of ERK1/2 signaling pathway protein.（2）The dsAAV9 carrying enhanced green fluorescent protein（eGFP）and dsAAV9-CaMEK vectors were transfected into neonatal rat cardiomyocytes,and the transfection efficiency of dsAAV9 vector was determined by fluorescence microscopy and Western blot.Western blot was used to evaluate the effect of CaMEK gene overexpression on ERK1/2 signaling pathway.Hypoxia/reoxygenation model and oxidative stress model were established.The main indicators were cardiomyocyte apoptosis,mitochondrial membrane potential（Δψm）,key proteins of ERK1/2 signaling pathway and expression of autophagy-related proteins.（3）Eight weeks of ApoE^-/-male mice were fed with a high-fat diet for 4 months.dsAAV9-eGFP and dsAAV9-CaMEK viral vectors were injected through the tail vein,respectively.Five weeks after transduction,Langendorff ex vivo I/R and in vivo I/R model were established.The main indicators were myocardial infarct size,apoptosis,key proteins of ERK1/2 signaling pathway and expression of autophagy-related proteins.Cardiac ultrasound was performed to evaluate the cardiac function of mice with in vivo I/R model within 1 month after reperfusion,and masson staining was used to evaluate myocardial fibrosis after I/R injury.Result:（1）（1）Ischemic post-conditioning had no protective effects on the myocardium of AS mice,and there were no significant differences in myocardial infarction area,apoptosis and myocardial enzymes between I/R group and IPost group（P>0.05）.（2）Ischemic post-conditioning exerts myocardial protective effects by activating ERK1/2 and p70S6K in the myocardium of healthy mice,but IPost treatment cannot effectively activate the ERK1/2 pathway in the myocardium of AS mice.（2）（1）After transfection with dsAAV9 vectors for 5 days,GFP and MEK proteins were highly expressed in myocardial cells.（2）After myocardial cells in all groups were stimulated by H/R and H₂O₂,the apoptosis of cardiomyocytes in CaMEK group was significantly lower than that in the control group（P<0.01）,the mitochondrial membrane potential increased,the cell viability increased,and lactate dehydrogenase（LDH）fell.At the same time,the activation of ERK1/2 signaling pathway inhibits the excessive autophagy of cardiomyocytes.PD98059 can inhibit the action of CaMEK gene.（3）（1）In the ex vivo myocardial I/R injury model,CaMEK gene overexpression effectively protects myocardial I/R injury of AS mice.The myocardial infarct size of I/R in the empty virus group and CaMEK gene transduction group was 30.88±2.25%vs.21.09±1.58%（P<0.01）.CaMEK gene transduction can effectively activate the ERK1/2 signaling pathway of AS myocardium,effectively reduce the apoptosis of myocardium and inhibit the excessive autophagy effect after I/R injury（2）For in vivo I/R studies,overexpression of CaMEK gene is also effective in reducing myocardial infarct size and cardiomyocyte apoptosis rate.The myocardial infarction area of GFP group and CaMEK group were43.41±2.47%vs.27.47±1.90%（P<0.05）.Targeted expression of CaMEK gene reduced apoptosis and myocardial enzyme level in AS myocardium,and the difference was statistically significant（P<0.01）.In addition,CaMEK gene transduction can effectively activate the ERK1/2-p70S6K-GSK3βsignaling pathway in myocardium of AS after I/R and inhibit the autophagy level after I/R injury.（3）Compared with the GFP group,the CaMEK group could reduce the left ventricular end-systolic area（LVESA）and leftventricular end-systolic volume（LVESV）values of the myocardium and increase the ejection fraction（EF）and fractional area change（FAC）values within 1 month after I/R,and the difference was statistically significant（P<0.05）.Conclusion:（1）Ischemic postconditioning adaptation does not play a protective role in myocardial I/R in AS mice and is associated with no further activation of the ERK1/2 signaling pathway.（2）The exogenous transfection of CaMEK gene can inhibit the autophagy level of cardiomyocytes by activating the ERK1/2 signaling pathway,reduce the apoptosis of cardiomyocytes caused by H/R and H₂O₂,stabilize the mitochondrial function,improve the activity of cardiomyocytes,and thus reduce the damage caused by H/R and oxidative stress to cardiomyocytes.（3）Targeted activation of myocardial ERK1/2 signaling pathway can prevent and treat AS myocardial I/R injury eventually by inhibiting autophagy,stabilizing mitochondrial function,reducing apoptosis,reducing infarct area,improving long-term cardiac function and other effects.CaMEK gene targeted transduction is expected to be an effective means for myocardial AS to fight ischemia reperfusion injury.