Research On The Mechanism Of AGE/RAGE Signal Involved In The Electrical Remodeling Of Diabetic Cardiomyocytes

Objective:The accumulation of advanced glycation end products（AGE）is an important factor in the occurrence and development of diabetic complications.The increase of AGE can lead to changes in the expression and function of ion channels in cardiomyocytes,and the abnormal function of ion channel proteins is the molecular basis for the occurrence of arrhythmia.Our previous study found that the expression of Cav1.2 and Kv4.3 in diabetic ventricular myocytes was down-regulated.In addition,the activity of Wnt/β-catenin and AKT/m TOR pathways also changed in diabetes,and may be related to arrhythmia,but the mechanism is unknown.This article intends to explore whether AGE can influence the function of myocardial ion channels by regulating the activity of Wnt/β-catenin or AKT/m TOR pathway,thereby inducing arrhythmia.Methods:Take 7-week-old ZL and ZDF rats with 8 each,and feed them with Purina5008feed for 1 week and then continuously feed the two groups of rats to 24 weeks of age to establish type 2 diabetic rat models;in primary SD rats cardiomyocytes were cultured for 48hours with 200μg/m L AGE reagent to construct AGE-damaged cell models,which were treated and divided into different groups according to the experimental purpose.After the completion of the model building,the following experiments were carried out:（1）The blood glucose and body weight of the two groups of rats were measured at the age of 10,13,and 17 weeks;（2）The heart function indexes of ZL and ZDF rats were detected by ultrasound of small animals;（3）WGA staining measures the surface area of ZL and ZDF rat ventricular myocytes;（4）Using whole-cell patch clamp technique to record the action potential,I_toand I_Ca-Lcurrent density,activation/inactivation,and resurrection curves of ZL and ZDF rat ventricular myocytes;（5）Western Blot detects the expression levels of the following proteins in ventricular tissues and cardiomyocytes:hypertrophy-related proteins ANP andβ-MHC;advanced glycation end products AGE and its receptor RAGE;Cav1.2and Kv4.3 ion channel proteins;Wnt/β-catenin pathway related proteins Wnt3a,β-catenin and nuclearβ-catenin;AKT/m TOR pathway related proteins AKT and its phosphorylated protein,m TOR and its phosphorylated protein.Results:1.In animal models,compared with ZL rats,ZDF rats have the following changes:（1）Blood sugar and body weight levels are significantly increased;（2）Small animal ultrasound results show that the diastolic function of the heart is weakened and the contraction function is enhanced;（3）WGA staining showed that the surface area of ventricular myocytes increased;（4）The action potential time course of ventricular myocytes was prolonged,but the overshoot value did not change significantly;the instantaneous outward potassium current I_toand the L-type calcium current I_Ca-Lcurrent density decreased,but There was no significant change in activation/inactivation and resuscitation kinetics;（5）The expression of cardiac tissue hypertrophy-related proteins ANP andβ-MHC was up-regulated;the expression of AGE and RAGE was up-regulated;the expression of Kv4.3 and Cav1.2 channel proteins was down-regulated;The expressions of Wnt/β-catenin pathway related proteins Wnt3a,β-catenin and nuclearβ-catenin were all up-regulated;the expressions of AKT/m TOR pathway related proteins AKT and m TOR did not change significantly,but the expressions of phosphorylated proteins p-AKT and p-m TOR were down-regulated;2.In the cell model,it was found that:（1）AGE can induce the down-regulation of Cav1.2 and Kv4.3 channel proteins,the Wnt/β-catenin pathway is activated,the AKT/m TOR pathway is down-regulated,and anti-RAGE can reverse its effect;（2）Theβ-catenin inhibitor XAV939 can block the down-regulation of Cav1.2 and Kv4.3 channel proteins induced by AGE;（3）PI3K and AKT inhibitors can directly down-regulate the expression of Cav1.2 and Kv4.3 channel proteins.Conclusion:This study found that AGE can activate the Wnt/β-catenin pathway in diabetes,while inhibiting the AKT/m TOR pathway,down-regulating the expression of Cav1.2 and Kv4.3 ion channel proteins,leading to electrical remodeling of cardiomyocytes,suggesting that Wnt/β-catenin or/And AKT/m TOR signaling pathway may be a potential target for the treatment of diabetic ventricular arrhythmia.