Mechanism Of Fatty Acid Mediated Mitochondrial Reactive Oxygen Species In Myocardial Injury Induced By Severe Hypoglycemia In Diabetes

Background and Objective:Lots of clinical studies have shown that severe hypoglycemia is closely related to the increase of cardiovascular events in diabetic patients.However,how severe hypoglycemia leads to cardiovascular events,and its specific mechanism remains unclear.During hypoglycemia,the massive release of catecholamine（mainly adrenaline）considered to be the main cause of cardiovascular events.Then the heart is an organ highly dependents on fatty acids as energy substrates.In diabetes,elevated free fatty acids in the blood will further promote the fatty acid metabolism.It is not clear that whether the increased fatty acid metabolism in the heart of diabetes would affect the cardiac reaction to catecholamine during hypoglycemia.Mitochondria are the main organelles producing reactive oxygen species（ROS）.Both increased fatty acid metabolism and myocardialβ-adrenoceptor activation are accompanied by production of mitochondrial ROS（mt ROS）.When excessive ROS accumulates in cell,it will damage cardiomyocytes and induce cardiac dysfunction.Therefore,it is worth further exploring whether the mt ROS accompanied by the enhancement of cardiacβoxidation of fatty acid in the diabetes could promote the occurrence of cardiovascular events after severe hypoglycemia.The aim of this study is to observe the effects of severe hypoglycemia on cardiac function and myocardial metabolism in mice with or without diabetes.In order to explore its differential effects,the effects of high fatty acid pretreatment on cardiomyocytes afterβ-adrenoceptor activation and the role of mt ROS in the above in vivo and in vitro models are further clarified.To provide potential measures for clinical diabetic patients to prevent cardiovascular events after SH.Materials and methods:Firstly,male C57BL/6J mice were divided into four groups,including non-diabetic control group,non-diabetes+severe hypoglycemia group,diabetes group and diabetes+severe hypoglycemia group.Mice were first given intraperitoneal injection of streptozotocin to form a diabetic state.Subsequently,diabetic mice received severe hypoglycemic intervention for one time.At the end of experiment,the cardiac systolic function,serum cardiac troponin I（c Tn I）and the expression of cardiac natriuretic peptide and brain natriuretic peptide were detected by color Doppler ultrasound,enzyme-linked immunosorbent assay（ELISA）,real-time quantitative polymerase chain reaction（RT-q PCR）to evaluate the cardiac function.Hematoxylin eosin（HE）staining was used to observed the cardiac structure.The levels of cardiac inflammation and oxidative stress were detected by ELISA.The contents of triglyceride and ceramide in myocardial tissue and serum free fatty acids were detected by oil red O staining and ELISA.RT-q PCR and western blot（WB）were used to detect the expression of key proteins of lipid metabolism and glucose uptake in myocardial tissue.Then,cardiomyocytes pretreated with or without high fatty acid were treated withβreceptor agonist（isoproterenol）in vitro.After the experiment,cell counting kit,immunofluorescence staining and hippocampal energy metabolism instrument were used to evaluate cell activity,mitochondrial morphology and function.The levels of ROS and（calcium ion）Ca²⁺from mitochondria to cytoplasm,apoptosis indexes and the expression of mitochondrial related apoptosis proteins were evaluated by immunofluorescence staining and WB.After administration of mitochondrial ROS scavenger（Mito-TEMPO）during pretreatment with high fatty acid of cardiomyocytes,the above indexes were detected again to evaluate the role of mitochondrial ROS in it.Finally,diabetic mice pretreated with Mito-TEMPO,and then severe hypoglycemia intervention was performed again.The cardiac function,key proteins of myocardial metabolism,mitochondrial membrane potential and the content of myocardial adenosine triphosphate（ATP）were evaluated again by color Doppler ultrasound,ELISA and WB.Results:After experiencing severe hypoglycemia of diabetic mice,the systolic function of the heart was decreased,serum c Tn I was increased,and cardiac structure disorder aggravated.Then,the inflammatory factors involved tumor necrosis factor-α（TNF-α）and interleukin-1β（IL-1β）further increased,the ROS further accumulated with antioxidant factor glutathione（GSH）further decreased.We also observed that,the deposition of lipid with increased triglyceride content in the myocardium,and the elevated free fatty acids.In addition to that,the expression of myocardial lipid metabolism related proteins including cluster of differentiation 36（CD36）,fatty acid transporter 1（FATP-1）,carnitine palmityl transferase1（CPT-1）,fatty acyl coenzyme A synthetases（FACS）,medium-chainacyl-Co A dehydrogenase（MACD）and glucose transporter 4（GLUT4）decreased,after diabetic mice subjected to severe hypoglycemia.Although it was accompanied by the increase of myocardial TNF-α,interleukin-6（IL-6）,interleukin-18（IL-18）and oxidation factor ROS,and the decrease of antioxidant factor GSH,following the exposure to severe hypoglycemia in non-diabetic mice.There were no significant changes in cardiac function and structure.In addition to that,lipid deposition and triglyceride accumulation were not observed.On the contrary,the expression of key proteins of myocardial lipid metabolism were increased.In vitro results were further found that cardiomyocytes pretreated with high fatty acid were more vulnerable to be damaged afterβ-adrenoceptor activation,which was characterized by decreased cell viability,disordered mitochondrial structure,dissipation of mitochondrial membrane potential,decreased mitochondrial oxidative phosphorylation and non-apoptotic injury.It also accompanied by the accumulation of ROS and calcium from mitochondria to cytoplasm.Elimination of mt ROS in high fatty acid environment,and thenβ-adrenoceptor agonist was administrated to cardiomyocytes,the changes of the above indexes showed different degrees of reversal.Then,scavenging mitochondrial ROS in diabetic mice could increase the cardiac systolic function,and reduce the level of c Tn I in diabetic mice after severe hypoglycemia.In addition,lowering the level of mt ROS in the diabetic mice could effectively reverse the reduction of the key protein of cardiac lipid metabolism including CD36,FATP1,CPT-1,FACS,MCAD and GLUT4 after severe hypoglycemia.Mito-TEMPO also increase the mitochondrial membrane potential and ATP content of myocardial tissue after severe hypoglycemia in diabetic mice.Conclusions:SH aggravated cardiac dysfunction and inhibited myocardial metabolism in diabetic mice,but did not affect cardiac function in non-diabetic mice,and promoted myocardial metabolism on the contrary.Cardiomyocytes pretreated with high fatty acids were more susceptible toβ-adrenoceptor activation damage and mitochondrial ROS plays a key role in this process.This might explain the cardiac differential outcome of severe hypoglycemia in diabetic mice.The elimination of mitochondrial ROS in diabetic mice could effectively prevent cardiac dysfunction and metabolism inhibition induced by SH.Providing a potential target for prevention of cardiovascular events after SH in diabetic patients.