Proteome Research On The Increased Vulnerability Of Myocardial Ischemia/Reperfusion Caused By Obesity In Mice

Objective:Obesity was regarded as an impact which could increase the incidence and severity of myocardial ischemia/reperfusion injury,but the mechanism is still unclear.For the increase of myocardial ischemia/reperfusion vulnerability,we should not only focus on the process of ischemia/reperfusion injury,but also study the pathophysiological changes of myocardium caused by obesity,which maybe an important basis for the decrease of tolerance to ischemia/reperfusion.Therefore,to preliminarily explore the mechanism of myocardial ischemia/reperfusion vulnerability,we used proteomics to analyze the differential protein expression between obese mice and normal mice.Methods:Firstly,obesity model of mice was established by high-fat feeding,and performed myocardial ischemia/reperfusion.To determine whether obesity increased the vulnerability of myocardial ischemia/reperfusion,we compared with the differences of cardiac function,myocardial infarction area and fibrosis degree between obese mice and normal mice.Then,we used myocardial tissues of obese and normal mice without ischemia/reperfusion treatment for proteomic analysis to screen the differentially expressed proteins which aimed to explore the potential molecular mechanism for the vulnerability of myocardial ischemia/reperfusion in obese mice.The main methods were as follows:1.C57/BL6 mice were fed with high fat diet for 6 months to induce obesity mouse model;2.Regular monitoring the basic indicators of mice such as body weight,blood glucose,blood lipids;3.To establish the myocardial ischemia/reperfusion injury model in mice,and the cardiac function of the mice was measured by ultrasonic apparatus;4.Myocardial infarct areas were manifested by TTC staining;5.Masson staining was used to observe the deposition of collagen;6.Using proteomic techniques to analysis the myocardium tissues of normal and obese mice,potential targets were found through bio-informatics analysis;7.Western blot was used to confirm the proteomic results.Results:1.The overall average body weight of obese mice induced by high-fat diet for 6months was more than percentage of 60 comparing with normal mice,what`s more,the total cholesterol and blood glucose were significantly higher on obese mice,which indicated that the model was successfully and stably established;2.Echocardiographic results showed that there were no differences between normal mice and obese mice in cardiac function manifested by ejection fraction(71%)and shortening scores(35%).However,the cardiac function of obese mice had significantly decreased after suffering cardiac ischemia/reperfusion surgery.In normal mice,ejection fraction decreased 58% and short axis shortening decreased 28%,while ejection fraction decreased 47% and short axis shortening decreased by 20% in obesity mice;3.TTC staining results showed that the area of myocardial infarction in obese mice(41%)was significantly larger than normal mice(23%);4.Masson staining showed that there was percentage of 5 collagen in the normal group,while percentage of 7 in the obese mice myocardium.The collagen deposition in obese mice were observed more obviously after myocardial ischemia/reperfusion surgery,respectively percentage of 20,14;5.TMT proteomic techniques were used to analyze the myocardial tissues of normal and obesity mice.Comparing with normal mice,there were 45down-regulated proteins and 114 up-regulated proteins in obese mice,GO analysis showed that most differentially expressed proteins were related to fatty acid catabolism.And KEGG pathway analysis revealed that the differentially expressed proteins were mainly enriched in fatty acid degradation and PPAR pathway;Among the down-regulated differentially expressed proteins,AKT1 was correlated with the results of GO and KEGG pathway analysis.Western blot was used to verify the expression level of AKT1.The results showed that the protein level of AKT1 in obese mice was significantly downregulated compared with normal mice,which consistent with the results of proteomics.Conclusion:Obesity increases the susceptibility to myocardial ischemia/reperfusion injury,which may relate to the abnormal metabolism of fatty acids and glucose caused by obesity,and the down-regulation of AKT1 maybe an important factor in the vulnerability.