Study Of The Mechanism Of CAPN2 Regulating Hypoxia-Induced Autophagy In Cardiomyocytes Through PI3K/AKT/mTOR Pathway

Background:Myocardial infarction(MI)is a very threatening disease,which can lead to arrhythmia,cardiac arrest,and even sudden death.Persistent myocardial ischemia and hypoxia is the main cause of myocardial infarction.After ischemia and hypoxia,myocardial cells are often damaged,and severely damaged myocardial cells will start cell death programs,such as: Autophagy,Necrosis,Apoptosis,Ferroptosis,Pyroptosis,etc.Autophagy is considered to be a conserved process for the overall degradation and recycling of cytoplasmic components,which plays an important role in maintaining the homeostasis of tissues and cells.However,some studies have shown that autophagy beyond a certain threshold may be harmful and will promote cell death.Severe ischemia and hypoxia can lead to severe autophagy in the myocardium,and this abnormal severe autophagy will accelerate the damage of the corresponding myocardial cells,thereby promoting the formation of myocardial infarction.calpain(CAPN)is a neutral cysteine endopeptidase in cells.The most common calpain is u-calpain(CAPN1),m-calpain(CAPN2),and calpain 3(CAPN3).Among them,CAPN1 and CAPN2 were ubiquitously expressed in all tissues,while CAPN3 was only specifically expressed in skeletal muscle.It has been found that the concentration of Ca2+ in cardiomyocytes is significantly increased after ischemia/hypoxia and ischemia/reperfusion,which leads to the activation of CAPN2.By consulting the GEO database,it was found that the expression of CAPN2 increased especially significantly after myocardial ischemia and hypoxia,and basically had a trend of increasing with the extension of ischemia and hypoxia time.Studies have shown that the occurrence of ischemia and hypoxia and ischemia-reperfusion injury is accompanied by a significant enhancement of myocardial cell autophagy.These results suggest that CAPN2 may be involved in the occurrence and development of autophagy in myocardial infarction.CAPN2 or its CAPN family can promote autophagy in osteosarcoma cells,nerve cells,and liver cells.However,the effect of CAPN2 on cardiomyocytes in heart has not been reported.It has been reported that PI3K/AKT/mTOR pathway is an important pathway to regulate autophagy,but whether PI3K/AKT/mTOR signaling pathway is the pathway by which CAPN2 affects autophagy in hypoxic cardiomyocytes remains to be further explored by us.Research Objectives:1.To explore the expression characteristics of CAPN2 and multiple autophagy markers(Beclin1、ATG5、P62)in myocardial infarction;2.To explore the effect of CAPN2 on autophagy in cardiomyocytes after hypoxia;3.To explore whether CAPN2 affects cardiomyocyte autophagy through PI3K/AKT/mTOR pathway after hypoxia in cardiomyocytes.Research Methods:1.Expression characteristics of CAPN2 and multiple autophagy markers(Beclin1、ATG5、P62)in myocardial infarction tissue Western Blot and GEO database were used to detect the expression characteristics of CAPN2 and a variety of autophagy indicators(Beclin1、ATG5、P62)in myocardial infarction tissues2.Effects of CAPN2 on autophagy in cardiomyocytes after hypoxia(1)After cultured for 2 days,the cardiomyocytes grew to the logarithmic phase,and the confluence degree reached about 80%.The cells were treated with hypoxia for 0,12,18,24,36 and 48 hours,and the hypoxic cells were analyzed by Western blot.According to the expression of CAPN2 and autophagy indicators(Beclin1,Atg5,and P62),24 hours was finally selected as the hypoxia condition.(2)Cardiomyocytes were treated with hypoxia for 24 Hrs and transfected with si-CAPN2si-CAPN2 was transfected into cardiomyocytes,and the success of transfection was verified by q RT-PCR and Western blot.The protein levels of Beclin1,Atg5,and P62 were detected by Western blot.(3)Cardiomyocytes were treated with hypoxia for 24 Hrs and exogenous CAPN2 inhibitor MDL28170.The inhibitor MDL28170 was added to culture cardiomyocytes,and 10μmol/L was selected as the experimental condition in this study according to the proliferation ability of cardiomyocytes and the inhibition intensity of CAPN2 after adding the inhibitor MDL28170.It is meaningful to verify the inhibitory effect by Western blot.The protein levels of Beclin1,Atg5,and P62 were detected in cardiomyocytes of each group.3.CAPN2 affects cardiomyocyte autophagy through PI3K/AKT/mTOR pathway(1)Myocardial cells were treated with hypoxia for 24 Hrs and transfected with si-CAPN2,and the expression changes of related molecules in PI3K/AKT/mTOR signaling pathway were detected by Western blot.(2)The expressions of related molecules in PI3K/AKT/mTOR signaling pathway were detected by Western blot after the cells were exposed to hypoxia for 24 Hrs and the CAPN2 inhibitor MDL28170 was added.Results of the study:1.The expression of CAPN2 is increased and autophagy is enhanced after myocardial infarction.2.Under normoxic condition,silencing or inhibiting CAPN2 did not affect cardiomyocyte autophagy.Hypoxia increased the expression of CAPN2 and autophagy in cardiomyocytes.Compared with the hypoxia group,the expression of CAPN2 was decreased after hypoxia combined with silencing or hypoxia combined with inhibition of CAPN2,and at the same time,autophagy was weakened.3.Under normoxic condition,silencing or inhibiting CAPN2,PI3K/AKT/mTOR pathway protein expression had no significant change;After hypoxia treatment,the expression of CAPN2 increased,and the relative expression levels of p-PI3 K,p-AKT,and p-mTOR molecules decreased.Compared with the hypoxia group,the expression of CAPN2 was decreased,and the relative expression levels of p-PI3 K,p-AKT and p-mTOR molecules were increased after hypoxia and silencing CAPN2 or hypoxia and inhibiting CAPN2.Research conclusions:The present study suggests that CAPN2 may regulate autophagy in hypoxic cardiomyocytes through the PI3K/AKT/mTOR signaling pathway.This suggests that CAPN2 may be a potential target for the treatment or prevention of myocardial infarction.