The Exploration Of The Role And Mechanism Of Tom70 In Diabetic Cardiomyopathy

Research background and purpose:Diabetic Cardiomyopathy(DCM)is independent of coronary artery disease,and is an important complication that endangers the health and life of patients with diabetes mellitus(DM).According to relevant data from the World Health Organization,there are currently more than 400 million DM patients worldwide,22% of DM patients have heart failure,and about 3.3% of DM patients gradually develop heart failure each year.In the future,DCM will cause 50% of DM patients to die.The occurrence and development of DCM involves a variety of mechanisms,including mitochondrial dysfunction,increased oxidative stress,impaired myocardial insulin metabolism signals,elevated advanced glycation end products,inflammation,cardiac autonomic neuropathy,and microvascular dysfunction.Impaired mitochondrial function is considered to be the core link of the above-mentioned pathway activation and persistence.In the pathological state of DM,NADPH oxidase is over-activated,and the mitochondrial respiratory chain generates a large amount of reactive oxygen species(ROS).The accumulation of a large amount of ROS can destroy the redox homeostasis in the body,further aggravate mitochondrial dysfunction and form a vicious cycle to promote the occurrence and development of DCM.In addition,the work of normal myocardium relies on mitochondrial ATP production capacity.However,under high glucose conditions,it can induce the destruction of ATP synthase in myocardial cells,the production of mitochondrial superoxide and excessive apoptosis of myocardial cells,leading to myocardial remodeling and cardiac dysfunction.Mitochondria are organelles of eukaryotic cells which plays an important role in energy metabolism through oxidative phosphorylation.The mitochondria serves as the central roles for most of the mitochondrial precursor proteins that have to be imported into the organelle from the cytosol,and the mitochondrial outer membrane converting enzyme(Tom)complex has a preliminary recognition effect on preproteins in the cytoplasm.Among them,the mitochondrial outer membrane transformation complex subunit 70(Tom70)is one of the main receptor proteins of the Tom complex and it recognizes a large number of metabolisms.The primary input receptor for precursors ofbiomaterial carriers(such as ATP?ADP or phosphate carriers).Related research shows that Tom70 plays an important role in improving anemia,delaying the progression of Alzheimer's disease,treating hypertrophic cardiomyopathy and ischemic cardiomyopathy.The role of Tom70 in recognizing precursor proteins is regulated by metabolic signal switches.Under high glucose conditions,cAMP-dependent protein kinase(PKA)can phosphorylate Tom70 and directly regulate its input receptors,thereby regulating metabolism.Effect of product carrier input rate.Other research groups have confirmed that inhibiting Tom70 can aggravate myocardial mitochondrial damage and produce excessive ROS,thereby aggravating the reperfusion injury of ischemic cardiomyopathy.Therefore,we proposed that under the condition of high glucose,the phosphorylation level and expression of Tom70 decreased,and this change aggravated mitochondrial damage,increased the level of oxidative stress,and promoted the occurrence and development of DCM.Based on the above theoretical hypothesis,leptin receptor knockout(db / db)mice and corresponding wild type(WT)mice to construct a mouse cardiac cell model with high glucose and lipid.At the same time,combined with molecular biology technology and morphological organization technology,the mitochondrial morphology and function evaluation will be conducted at the subcellular level.Carry out related research work using small animal cardiac ultrasound to evaluate the structure and function of the heart.It is intended to clarify the role of Tom70 in DCM and preliminary explore related mechanisms in order to provide new intervention targets for DCM treatment and prevention.Materials and Methods:In this experiment,Tom70 siRNA interference technology was used to inhibit Tom70 expression and lentivirus interfered with over-expression of Tom70 to build related cell experiments and animal experimental models.In the cell experiments,Cell cytometry was used to measure Annexin V and propidium iodide(PI)double positive apoptotic cells;Apoptosis of cardiomyocytes was determined by the Annexin V-FITC Apoptosis Detection Kit;Western blot(WB)was used to detect the expression of apoptosis-related proteins Caspase3 and Caspase9;Dihydroethidine(DHE)was used toevaluate generation of superoxide anion oxygen radicals;Reactive oxygen species(ROS)generation was detected by an ELISA kit;Mitochondrial membrane potential was evaluated by using JC-1 kit;The ATP content in cardiomyocytes was measured by using a firefly luciferase-based ATP assay kit.And in animal experiment,a blood glucose meter was used to measure fasting blood glucose,ELISA kit was used to detect plasma insulin levels,Immunohistochemistry and Western blot were used to detect Tom70 expression,Real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR)technology was used to detect Tom70,ANP,BNP transcription,H&E staining and Wheat germ agglutinin(WGA)staining were used to detect the myocardial tissue morphology.Cardiac function was assessed by echocardiography,echocardiographic analysis of the early mitral diastolic wave/late mitral diastolic wave(E/A)ratio was used to reflect left ventricular diastolic function,left ventricular fractional shortening(LVFS)and left ventricular ejection fraction(LVEF)were used to assess left ventricular systolic function;mitochondrial morphologies detected by transmission electron microscopy.Result:1.Tom70 expression is inhibited in diabetic cardiomyopathyCompared with WT mice,fasting blood glucose and plasma insulin levels in db/db mice were significantly increased,while insulin sensitivity was significantly decreased,the average cardiac weight increased,the average cross-sectional area of myocardial cells increased,and myocardial fibrosis increased(P <0.01).At the same time,the Tom70 gene transcription and the protein expression level decreased significantly(P<0.01).2.Inhibition of Tom70 expression can aggravate diabetic cardiomyopathyCompared with the mice of WT with T siRNA,myocardial hypertrophy and myocardial fibrosis in the db/db(T siRNA)group were significantly increased,ANP and BNP transcription levels were significantly increased,and LVEF,LVFS,and E/A ratio were significantly reduced(P <0.01);compared with mice of db/db with scramble RNA(T scRNA),the db/db(T siRNA)group can further aggravate the above indicators(P <0.01).3.Downregulation of Tom70 aggravates HGHF-mediated oxidative stress injuryand apoptosis in cardiomyocytesCompared with the cells in normal medium with Tom70 siRNA,the HG-HF(T siRNA)group significantly increased the apoptosis level and the expression of apoptosis-related molecules Caspase3 and Caspase9,the levels of superoxide anion and ROS were significantly increased,and mitochondrial function was significantly impaired(P <0.01).Compared with the cells in HG-HF with Tom70 siRNA,the apoptosis,oxidative stress and mitochondrial function of the HG-HF(T scRNA)group were significantly improved(P <0.01).3.Overexpression of Tom70 reduces HGHF-induced cardiomyocyte apoptosis and ameliorate diabetic cardiomyopathyIn the cell experiment part,compared with the cells in normal medium treated with the same lentivirus,the HG-HF(Tom70)group had higher apoptosis and apoptosis-related molecules(P<0.05,P<0.01);compared with the cells in HG-HF medium infected with lentivirus expressing GFP alone,after being transfected with Tom70,lentivirus can significantly improve apoptosis(P<0.01).In the animal experiment part,compared with the mice of WT with lentivirus expressing GFP alone,the mitochondrial morphology was destroyed,mitochondrial ATP and MMP levels were significantly suppressed,the myocardial apoptosis was increased,myocardial hypertrophy and myocardial fibrosis increased,and LVEF And LVFS were significantly reduced(P<0.01),and overexpression of Tom70 could significantly improve the above conditions in db / db mice(P <0.05,P <0.01).Conclusion:1.The expression level of Tom70 in the heart of db/db mice is reduced.Inhibiting the expression of Tom70 aggravates the mitochondrial dysfunction,oxidative stress and apoptosis of HG-HF mediators,thereby further aggravating DCM.2.Overexpression of Tom70 can reduce the level of oxidative stress and apoptosis,repair mitochondrial morphology and function,and thus play a role in improving DCM.