| BackgroundIn the past years,the number of people with diabetes mellitus has been growing rapidly around the world.China has a massive population of diabetes.According to the study conducted by Shanghai Institute of Endocrinology and Metabolism and Chinese Center for Disease Control and Prevention,the morbidity rate of adult diabetes in China has risen to 11.6%.As is estimated,the risk of suffering from cardiovascular diseases for diabetic patients is 2 to 3 times the risk for non-diabetic individuals.Cardiovascular diseases are the leading cause of death in people with diabetes mellitus,of which ischemic myocardial infarction is the most critical threat.Myocardial ischemia-reperfusion induces cardiomyocyte necrosis,cardiomyocyte apoptosis and ventricular remodeling,which are the main pathophysiological fundamental of cardiac insufficiency after reperfusion therapy to cure myocardial infarction.It is reported that post-myocardial infarction fatality rates for diabetic patients is nearly twice the rate for people without diabetes mellitus,suggesting that diabetic heart suffers exacerbated impairment after ischemic injury.But the mechanisms underlying the susceptibility of diabetic heart remains undefined.Investigating mechanisms behind the susceptibility of diabetic heart to ischemic injury is significant for clinical treatment and prevention of ischemic heart disease with diabetes mellitus.Necrosis and apoptosis of cardiac myocytes are important pathological pathways involved in the development of heart disease.Cardiomyocyte apoptosis has been regarded as a mode of programmed death in the past,and myocardial necrosis is considered to be a passive cell event and difficult to control effectively.Necrosis is the main morphological manifestation of cell death caused by myocardial ischemia,and necrosis also causes severe inflammatory response,causing damage cascade amplification.The harm of necrosis to the myocardium is more intense,and its research deserves more attention.Programmed necrosis(necroptosis),a caspase independent cell programmed death pattern mediated by death receptor,has made a significant breakthrough in the theory of cell death.The discovery and confirmation of necroptosis and its regulatory pathways make it possible to regulate necrosis.Recent studies have shown that myocardial necroptosis plays an important role in some severe cardiac pathological conditions like myocardial infarction,ischemia/reperfusion injury and heart failure.RIP3 and Ca MKII are key proteins involved in the pathway of myocardial necroptosis.RIP3 directly phosphorylates Ca MKII while indirectly oxidizing Ca MKII by ROS generation to activate myocardial necroptosis.Therefore,the activation state of the RIP3/Ca MKII signaling pathway is closely related to the MI/R damage.But now,in the process of myocardial MI/R injury in diabetes,the severity of myocardial programmed necrosis;activation of the RIP3-Ca MKII pathway;whether the signaling mechanism in diabetic myocardial ischemia increased vulnerability and other important issues need to be further elucidated.Based on the consideration above,this subject aims at observing the differences and changes of RIP3/Ca MKII signaling pathway in normal and diabetic myocardium during MI/R from in vivo and in vitro animal experiments,exploring the new mechanism of diabetic myocardial ischemia damage,providing therapeutic targets and new experimental basis for clinical treatment of diabetic patients with ischemic heart disease.Aims 1.To determine whether myocardial necroptosis is involved in the ischemic susceptibility of diabetic myocardium;2.To investigate the activation status of RIP3-Ca MKII pathway and the degree of myocardial necroptosis in diabetic myocardial I/R;3.To identify whether suppressing RIP3-Ca MKII pathway is effective to decrease myocardial necroptosis in diabetic myocardial I/R,protect cardiac function and increase survival.MethodsC57 mice were taken as the research object,and the diabetes model was induced by STZ.On this basis,the acute MI/R model of mice(ischemic 30 min / reperfusion 4h)was established.Myocardial infarct size was measured by Evans blue/TTC staining.Cardiac function was measured by echocardiology.Myocardial necroptosis was assessed by the ratio of Evans blue positive area and serum LDH concentration.The protein expression level of RIP3-Ca MKII signal and the level of phosphorylation / oxidation were detected by Western Blot method,and the activation degree of myocardial Ca MKII was determined.Adenovirus mediated RIP3-si RNA,Ca MKII inhibitor KN-93 and Ca MKII phosphorylation / oxidative site mutation were used to verify the effect of Ca MKII on necroptosis in diabetic ischemic myocardium.Results 1.Compared with the control group,a dose of 40mg/kg for 5 consecutive days intraperitoneal injection of STZ solution can effectively induce C57 mice diabetes: in the fifth week after injection,mice showed the characteristics of type 1 diabetes model.The body weight,heart weight,heart rate and heart weight / tibia length decreased significantly.Fasting blood glucose was higher than 10mmol/L and blood glucose of 60 minutes time point in IPGTT was higher than 15mmol/L,which confirmed the success of type 1 diabetes model establishment.2.By the measurement of myocardial Evans blue area and the level of serum LDH,Evans blue/TTC double staining method to measure myocardial infarct size and echocardiographic assessment of cardiac function,we confirmed that compared to thecontrol group,diabetic myocardial necroptosis after MI/R increased by 35%,the area of myocardial infarction was significantly increased,cardiac systolic and diastolic function decreased significantly 4 weeks after MI/R,the survival rate in 60 days was significantly lower(P<0.01).These results indicate that necroptosis caused by MI/R in diabetic status is significantly increased,and myocardial necroptosis is involved in the vulnerability of diabetic myocardium to ischemia.3.To investigate the mechanism of enhanced necroptosis in diabetic myocardial I/R,multiple reperfusion time points after iscemia for 30 minutes were set to examine phosphorylated Ca MKII and oxidized Ca MKII.The results showed that the level of Ca MKII oxidation in the diabetic group and the control group increased at the beginning of MI/R,reached the peak at 10 minutes after reperfusion,and returned to the level before reperfusion 1 hour later.However,Ca MKII oxidation peak level of MI/R myocardium in diabetic group was significantly 16% higher than the control group.30 minutes after the onset of reperfusion,Ca MKII phosphorylation level increased.More importantly,1 hour after reperfusion,diabetic MI/R myocardial Ca MKII phosphorylation reached the peak value,of which the peak level occurred earlier than that of the control group.The findings above suggest that the Ca MKII oxidation level of MI/R myocardium increases significantly in diabetic state while the activation of phosphorylated Ca MKII occurs earlier in diabetic state.The abnormal activation of time and intensity leads to increased myocardial necroptosis and worsened MI/R damage in diabetic myocardium.4.In order to investigate the role of Ca MKII signal in the process of myocardial necroptosis,we performed the mutation of Ca MKII oxidative site and phosphorylative site to achieve Ca MKII inhibition in vitro.The results showed that Ca MKII phosphorylation site mutation group,Ca MKII oxidative site mutation group and Ca MKII phosphorylation site / oxidation site double mutation group had higher cell survival rate after hypoxia/reoxygenation treatment than those in the control group.Therefore,our results indicate that phosphorylation and oxidation of Ca MKII both mediate myocardial necroptosis,and early inhibition of Ca MKII activation may be an effective protection of myocardium.5.Aiming at the advanced and enhanced abnormal activation of Ca MKII signal in diabetic MI/R myocardial necroptosis,out study compared the early(15 min before ischemia,intraperitoneal injection)and late(at the beginning of reperfusion,intraperitoneal injection)use of Ca MKII inhibitor KN-93 on myocardial Ca MKII activation in diabetic MI/R myocardial necroptosis.The results show that early administration of KN-93(15 minutes before ischemia)can effectively inhibit diabetic MI/R myocardial necroptosis,reduce myocardial infarction and improve the long-term survival rate;and the late administration of KN-93 after reperfusion can only reduce diabetic MI/R myocardial necroptosis in part,of which myocardial protective effect is weakened obviously.6.In order to elucidate the effect of RIP3 on the regulation of Ca MKII signal in diabetic myocardial I/R and the effect of necroptosis on myocardium,the expression of RIP3 in myocardium of I/R was detected,and the interaction between RIP3 and Ca MKII was detected by Co-IP.The results show that the myocardial RIP3 level in diabetic MI/R increased significantly compared with that in the control group(P<0.01),and the binding effect of RIP3 and Ca MKII in myocardium of diabetic I/R increased significantly.These results suggest that RIP3 may be a direct acting factor for the activation of Ca MKII in diabetic myocardium.7.To further investigate whether or not we can protect the myocardium of diabetic I/R by inhibiting RIP3 to suppress myocardial necroptosis,our research used the adenovirus packaging si RNA to knockdown RIP3(Ad-RIP3 si RNA)in diabetic myocardium in vivo,detected the level of ROS generation,myocardial necroptosis and the degree of Ca MKII phosphorylation and oxidation.The results show that myocardial injection of Ad-RIP3 si RNA 48 hours before I/R surgery can effectively reduce the level of RIP3 in diabetic myocardium;inhibition of RIP3 can effectively inhibit ROS in diabetic myocardial I/R,reduce Ca MKII oxidation and phosphorylation level,ultimately inhibit myocardial necroptosis,effectively suppress the degree of diabetic myocardial I/R injury and exert myocardial protective effectConclusions 1.This study confirms for the first time that the Ca MKII oxidation level of MI/Rmyocardium increases significantly in diabetic state,and Ca MKII phosphorylation is advanced earlier.It leads to the earlier advanced and enhanced activation of myocardial necroptosis,which exacerbates the MI/R damage of diabetic myocardium.2.RIP3 is an important regulator of abnormal activation of Ca MKII in diabetic myocardium.3.Aiming at RIP3-Ca MKII pathway to suppress myocardial necroptosis in diabetic myocardial I/R can effectively reduce the vulnerability of diabetic myocardium to ischemia and exert myocardial protection. |
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