The Mechanism Of MCU Mediates Abnormal Mitochondrial Calcium Uptake And Cardiomyocyte Apoptosis In High Glucose Environment

Background:As the number of diabetics worldwide continues to increase,diabetic cardiomyopathy has become one of the main causes of cardiovascular disease risk in diabetic patients.DCM is a disease associated with metabolic changes,mainly characterized by left ventricular myocardial contraction and diastolic dysfunction.Improper treatment can cause arrhythmia,cardiogenic shock,heart failure,and even severe death in severe cases.Currently,the pathophysiological mechanism of DCM has not been fully elucidated,but mitochondrial dysfunction caused by imbalanced mitochondrial calcium homeostasis is considered one of the important pathological mechanisms of DCM.MCU is the main channel for mitochondrial Ca²⁺uptake,playing an essential role in the regulation of mitochondrial calcium homeostasis.Whether it has participated in the occurrence and development of DCM is rarely reported at domestic and overseas.Our preliminary experiments found that the expression of MCU was reduced in the myocardial cells of diabetic mice.Whether changes in the expression of MCU at the cellular level affect mitochondrial function and thus participate in the occurrence of DCM have not been confirmed by studies.Therefore,this study aims to observe the effect of abnormal expression of MCU under high glucose induction on mitochondrial calcium uptake,to further explore the mechanism of high glucose-induced apoptosis of cultured cardiomyocytes,and provide a novel potential target for the pathogenesis and clinical treatment of DCM.Objective:1.Observe the effect of high glucose culture on MCU expression in H9C2 cardiomyocytes.2.To explore the effect of MCU expression changes on mitochondrial function and then the apoptosis of H9C2 cardiomyocytes and the mechanism.Method:1.Culture H9C2 cardiomyocytes in vitro.Cardiomyocytes were given high glucose?HG group,glucose concentration of 33mmol/L?and normal glucose?NG group,glucose concentration of 5.5mmol/L+27.5mmol/L mannitol?culture.Experiments were conducted after culturing for 72h.The MCU of cardiomyocytes were detected by fluorescence quantitative PCR and Western Blot.Western Blot method was used to detect the expression of myocardial apoptosis-related proteins cleaved caspase3,cleaved caspase9 and Bcl-2.The mitochondrial calcium dye Rhod-2 was used to detect the mitochondrial calcium uptake capacity of cardiomyocytes.The change of membrane potential was detected using JC-1 kit.The ATP detection kit was used to detect the mitochondrial ATP content.NADP⁺/NADPH detection kit and the GSH/GSSG detection kit were used to detect the antioxidant capacity of cardiomyocytes.The changes of ROS in cardiomyocytes were detected by Mito-SOX staining reagent.Flow cytometry and TUNEL kit were used to detect cardiomyocyte apoptosis.2.Culture H9C2 cardiomyocytes in vitro.Cardiomyocytes were cultured in normal sugar for 72h,and after interference with siRNA to down-regulate the expression of MCU,they were divided into normal group?NG group,glucose concentration of 5.5mmol/L?,MCU siRNA blank control group?NG+NC siRNA group?,and experimental group?NG+MCU siRNA group?with down-regulated MCU expression.The MCU was detected by fluorescence quantitative PCR and Western Blot method.Western Blot method was used to detect the expression of myocardial apoptosis-related proteins cleaved caspase3,cleaved caspase9 and Bcl-2.The mitochondrial calcium dye Rhod-2 was used to detect the mitochondrial calcium uptake capacity of cardiomyocytes.The change of membrane potential was detected using JC-1 kit.The ATP detection kit was used to detect the mitochondrial ATP content.NADP⁺/NADPH detection kit and the GSH/GSSG detection kit were used to detect the antioxidant capacity of cardiomyocytes.The changes of ROS in cardiomyocytes were detected by Mito-SOX staining reagent.Flow cytometry and TUNEL kit were used to detect cardiomyocyte apoptosis.Result:1.Compared with the normal group?NG?,MCU expression in H9C2 cardiomyocytes in the high glucose group?HG?was significantly reduced?P<0.001?,mitochondrial Ca²⁺uptake was reduced?P<0.001?,membrane potential levels were low?P<0.0001?,ATP production was reduced?P<0.001?,NADP⁺/NADPH increased?P<0.01?and GSH/GSSG decreased?P<0.05?.ROS production increases?P<0.001?and cell apoptosis increases?P<0.01?.2.Compared with the normal group?NG?,the MCU expression in the experimental group?NG+MCU siRNA?H9C2 cardiomyocytes was reduced?P<0.001?,mitochondrial Ca²⁺uptake was low?P<0.001?,membrane potential levels were low?P<0.0001?,ATP production was reduced?P<0.0001?,NADP⁺/NADPH increased?P<0.01?and GSH/GSSG decreased?P<0.05?.Increased ROS production?P<0.001?and increased apoptosis?P<0.01?.3.Compared with the control group?NG+NC siRNA?,the experimental group?NG+MCU siRNA?H9C2 cardiomyocytes had lower MCU expression?P<0.001?,less mitochondrial Ca²⁺uptake?P<0.0001?,lower membrane potential levels?P<0.001?,reduced ATP production?P<0.0001?,increased NADP⁺/NADPH?P<0.01?and decreased GSH/GSSG?P<0.05?.Increased ROS production?P<0.0001?and increased apoptosis?P<0.01?.4.Compared with the normal group?NG?,the control group?NG+NC siRNA?showed no difference in mitochondrial Ca²⁺uptake,membrane potential level,ATP production,NADP⁺/NADPH,GSH/GSSG,ROS production and apoptosis?P>0.05?.Conclusion:1.In a high-sugar environment,MCU expression of H9C2 cardiomyocytes decreases,and mitochondrial calcium uptake decreases.2.Decreased MCU expression causes mitochondrial dysfunction,increased ROS production,and enhanced oxidative stress.3.Decreased MCU expression causes increased cardiomyocyte apoptosis.