Role And Mechanism Of AMPK-CHMP2B In The Regulation Of Myocardial Autophagy

Background: There are 330 million patients with cardiovascular diseases in China,and cardiovascular disease is the leading cause of death among urban and rural residents.It is important to elucidate the pathogenesis and molecular mechanism of cardiovascular diseases.Autophagy is a lysosome dependent mass degradation system.Current studies have confirmed that autophagy is essential to maintain the normal function of cardiomyocytes.However,the mechanism and key molecules of autophagy regulation remain to be clarified.Charged Multivesicular Body Protein 2B(CHMP2B)is a key regulator of autophagic lysosomal vesicular fusion,and previous studies have shown that CHMP2 B plays an important role in various neurodegenerative changes.Our previous study found that during myocardial ischemia/reperfusion(MI/R)injury,excessive accumulation of CHMP2 B would lead to autophagy dysfunction and damage myocardial cells.Therefore,the search for the negative regulatory mechanism of myocardial CHMP2 B may be a new way to regulate myocardial autophagy.Adenosine 5’-monophosphate-activated protein kinase(AMPK)is an important myocardial protective factor.Existing studies have confirmed that AMPK is an important regulator of autophagy function of myocardial cells.However,with the deepening of research on autophagy,the detailed molecular mechanism of AMPK regulating myocardial autophagy is still being improved.The purpose of this study was to clarify whether AMPK regulates myocardial autophagy through CHMP2 B and to clarify the potential regulatory mechanisms.Methods: 1.Taking H9c2 cells as the research object,the autophagy of cardiomyocytes was detected by the intervention of AMPK inhibitor and AMPK agonist.2.Western blotting was used to detect the expression levels of p-AMPK,AMPK,CHMP2 B,autophagy marker proteins LC3,p62,LAMP1,Atg5 and Tubulin proteins.3.Bioinformatics analysis screened the potential transcription factors of CHMP2 B,and verified the effect of transcription factors on m RNA levels by q RT-PCR.4.CHIP and luciferase reporter gene system were used to determine the role of transcription factors.5.q RT-PCR was used to verify transcription factors and test nucleic acid expression levels.Result: 1.Adenovirus overexpression of CHMP2 B can significantly inhibit myocardial autophagy flux.Protein levels of Atg5,LC3 II/I,LAMP1,and p62 all increased synchronously,indicating impaired functional autophagy flux.2.Activation of AMPK can effectively improve myocardial autophagy flux;Inhibition of AMPK can effectively inhibit myocardial autophagy flux.3.The m RNA level of CHMP2 B was detected,and the activation of AMPK significantly down-regulated the m RNA level of CHMP2 B.Inhibition of AMPK led to up-regulation of CHMP2 B m RNA.It is suggested that AMPK can inhibit CHMP2 B transcription.4.The 13 potential transcription factors regulating CHMP2 B transcription were screened by bioinformatics analysis,and the transcription factors related to energy metabolism and autophagy were further screened and verified by RT-PCR.These results suggest that Fox K2 may be involved in AMPK transcriptional regulation of CHMP2 B.5.CHIP and luciferase reporter gene system confirmed that Fox K2 can bind to the CHMP2 B promoter region to promote CHMP2 B transcription.6.Nuclear/component analysis confirmed that activation of AMPK could inhibit Fox K2’s entry into the nucleus and inhibit its transcriptional activity.This study confirmed that up-regulation of CHMP2 B can lead to myocardial autophagy dysfunction.On this basis,this study found that activation of AMPK could inhibit the entry of Fox K2 into the nucleus,thus down-regulating the transcription level of CHMP2 B.The negative regulation of AMPK on CHMP2 B may be a new mechanism of myocardial autophagy regulation and provide a new idea for myocardial protection mechanism.