| Background Diabetes mellitusis(DM)is a serious threat to human health,and its incidence in the world is still increasing as well as the annual death of diabetic patients.China is confronted with the greatest challenge in the world in the battle of resisting DM.There are 110 million diabetics in China in 2015,and about one million people die from diabetes each year.Diabetes is the most important risk factor for cardiovascular disease(CVD),and cardiovascular complications are the leading cause of death in diabetics.Researches have shown that the incidence and mortality of CVD in patients with diabetes was significantly higher than those patients without diabetes.Previous studies have attributed this effect of diabetes to its hyperglycemia.However,clinical studies have shown that intensive glycemic control does not significantly improve cardiovascular complications in diabetic patients.Therefore,to explore the actualcardiovascular risk factors associated with diabetes,is extremely important for the control of cardiovascular complications and improve the lives of patients with diabetes.Branched-chain amino acids(BCAA)belong to essential amino acids and we obtain them only from food and drink.BCAA is the bricks that are indispensable for protein synthesis its regulation.In addition,BCAA is also involved in the regulation of cell proliferation,apoptosis,oxidative stress,autophagy and many other important life activities.As early as 1976,report published on the JCI had found that the plasma BCAA level in diabetics was significantly higher than non-diabetics.Recently,study also found that the plasma BCAA level was also increased in patients with heart failure,while reduction of the level of BCAA in mice with heart failure,slowed down the progress of the disease.These studies suggested that BCAA metabolic abnormalities may be an important cause of cardiovascular disease in diabetes.It bas been reportedthat the accumulation of BCAA and its metabolic intermediates reduced the function of antioxidant system and promoted neuronal apoptosis,leading to nervous system damage.In addition,BCAAs inhibit the activity of m TORC1,m TORC2 and Akt in hepatocytes and resultant cell apoptosis.Akt plays an important role in the growth of embryonic myocardium,over-load induced cardiac hypertrophy and heart failure.Another experiment has proved that inhibition of m TORC2 significantly promote the progress of mouse heart failure.Thus,we hypothesize that BCAA metabolic disorders may be the cause of increased cardiac damage in diabetic patients with cardiovascular disease,which may be the function of the m TORC2-Akt pathway or to its antioxidant system.Aims 1.To research the relationship between the disorders of BCAA catabolism and diabetes-related myocardial ischemia-reperfusion injury and to explore the underling mechanism.2.To elucidate the effect of catabolic dysfunction of branched chain amino acid(BCAA)on over-load-induced cardiomyocyte apoptosis and explore its mechanism.Methods 1.Branched-chain amino acid catabolic dysfunction contributes to diabetes-related aggravation of myocardial ischemia/reperfusion injury and the wnderling mechanism.1.1 T2 DM mice model were induced by high-fat diet(HFD)and intermittent intraperitoneal injection of low-dose streptozotocin(STZ).1.2 Glucose tolerance test(GTT)and insulin tolerance test(TTC)were used to detect insulin resistance of mice.1.3 To establish a model of myocardial ischemia/reperfusion in mice: the mice were anesthetized with isoflurane inhalation,the skin of precordial area and the intercostal muscle was opened,and left anterior descending coronary artery was ligated for 40 minutes and then released.1.4 Myocardial injection of adenovirus was applied to over-expressed the target gene specifically.1.5 The cardiac function of mice was detected by ultrasound.1.6 Evans blue / TTC staining was used to detect the infarct size of the heart after ischemia/reperfusion.1.7 BCAA Levels in plasma and tissue homogenates was detected by BCAA Quantitative Kit.1.8The production of ROS was detected by DHE staining.1.9 Cell apoptosis was examined by TUNNEL staining and caspase-3 activity assay kit.1.10 Myocardial mitochondrial damage after I/R was observed by transmission electron microscopy.1.11 The culture and treatment of H9C2 cell line.1.12 Simulated hypoxia/reoxygenation of H9C2 cells in vitro.2 The influence of branched-chain amino acid(BCAA)metabolic dysfunction on isoproterenol(ISO)-induced cardiomyocyte apoptosis 2.1 Primary cardiomyocytes of adult mice was isolated and cultured in vitro.2.2 Cardiomyocyte apoptosis was induced by isoproterenol(ISO).2.3 Cell viability was de by CCK-8 Kit.2.4 Cell apoptosis was examined by TUNNEL staining and caspase-3 activity kit.2.5 Western-blot was used to detect the abundance of the target protein.2.6 Using plasmid to overexpress the target gene.Results 1 BCAA catabolic dysfunctions contribute to diabetes-related aggravation of myocardial ischemia/reperfusion injury via via down-regulation of Mn SOD protein expression and promotion of oxidative stress.1.1 Expression of PP2 Cm protein in myocardium of diabetic mice was downregulated.1.2 Overexpression of PP2 Cm in diabetic heart by myocardial injection of adenovirus reduced the diabetes-associated aggravation of myocardial ischemia/reperfusion.1.3 Compared with wild type(WT)mice,the injury after MI/R in PP2 Cm knockout mice(PP2Cm-KO)was significantly increased.1.4 The BCAA catabolic in the heart of diabetic mice was dysfunctional and the levels of BCAA and BCKA in cardiac tissue were increased markedly as a result.1.5 With the drug intervention to improve the BCAA catabolism in diabetic mice,the myocardial ischemia/reperfusion injury was significantly alleviated.1.6 Restoration of the BCAA catabolism in PPCPm-KO mice by drug intervention abolished the accentuation of their I/R injury compared with WT mice.1.7 Compared with WT mice,the apoptosis of myocardial cells in PP2Cm-KO mice after ischemia-reperfusion was significantly increased.1.8 Compared with WT mice,the expression of Mn SOD protein in PP2Cm-KO mice was significantly decreased;and after MI/R,the ROS production in PP2Cm-KO mice was markedly higher than that in WT mice.1.9 When corrected the BCAA metabolism dysfunction in PP2Cm-KO mice with BT2,,the expression of Mn SOD protein in myocardium effectively restored;and after MI/R,the ROS production in treated PP2Cm-KO mice(PP2Cm-KO + BT2)was significantly reduced and the mitochondrial morphological damage was significantly alleviated compared with those have not been treated(PP2Cm-KO + Vehicle).1.10 BCKA incubation down-regulated the expression of Mn SOD protein in H9C2 cells,and resultantly,increased the production of ROS,promoted cell apoptosis and decreased the survival after hypoxia-reoxygenation.2 The influence of branched-chain amino acid(BCAA)metabolic dysfunction on isoproterenol(ISO)-induced cardiomyocyte apoptosis.2.1 BCKA treatment decreased cardiomyocytes survival in ISO-induced cell death.2.2 BCKA incubation increased cardiomyocytes apoptosis after isoproterenol treatment.2.3 BCKA inhibited activation of Akt via downregulation of m TORC2.2.4 Overexpression of rictor enhanced cell survival and inhibited apoptosis.Conclusions 1.The BCAA catabolism in the heart of type 2 diabetic mice was dysfunctioned,which was involved in diabetes-associated exacerbation of myocardial ischemia/reperfusion injury.The mechanism in which BCAA catabolic dysfunctions aggravated myocardial ischemia/reperfusion injury was: BCAA and its metabolic intermediates reduced the expression of myocardial antioxidant enzymes and increased oxidative stress and cell apoptosis.2.BCAA catabolic dysfunctions enhanced the sensitivity of cardiomyocytes toISO-induced apoptosis by inhibiting the activity of the m TORC2-Akt signaling axis. |
PDF Full Text Request