Inhibition Caspase-3-mediated Cleavage On Beclin-1 DXXD In H9C2 Increases Autophagy Levels And Reduces Apoptosis And Necrosis Levels During Reoxygenation

Objective The aim of of this study was to explore whether Beclin-1 is cleaved by Caspase-3 in the H9c2 cardiomyocytes after hypoxia-reoxygenation,specific cleavage sites,and the level of autophagy,apoptosis,and necrosis of H9c2 cells after hypoxia-reoxygenation after protection of the cleavage site of Beclin-1.Methods In this study,rat H9c2 cardiomyocytes were used as subjects to observe the protein expression of Beclin-1 and its fragments after hypoxia for 5 hours;then H9c2 cardiomyocytes were treated with Caspase-3 inhibitor(Ac-DEVD-CHO).After pretreatment(Caspase-3 Inhibition,Cp3I),hypoxia-reoxygenation was followed(hypoxia 5 hours reoxygenation 2 hours,H5R2).The protein expression of Beclin-1 and its fragments was compared between Cp3I+H5R2 group,H5R2 group,Cp3 I group and sham operation group.It is predicted that the cleavage site of Beclin-1 by Caspase-3 is located in the structure of aspartic acid--arbitrary amino acid--arquivalent amino acid--aspartate(DXXD)of No.147.based on literature and amino acid sequence alignment.The mutant Beclin-1(M-Beclin-1)which mutated the cleavage site and the unmutated Beclin-1(W-Beclin-1)were transfected into H9c2 cells,respectively before hypoxia for 5 hours and reoxygenation for 2 hours.The expression and distribution of Beclin-1 and its fragments were compared.Finally,flow cytometry was used to observe the changes in autophagy,apoptosis,and necrosis levels during reoxygenation.Result During the reoxygenation of rat H9c2 cardiomyocytes 5 hours after hypoxia,the Beclin-1 fragment was marked on Western Blot;the number of Beclin-1 fragments in the Cp3I+H5R2 group was less than that in the H5R2 group(p<0.001);In the M-Beclin-1 group,Beclin-1 was significantly less sheared than the W-Beclin-1 group(p<0.05).After respective overexpressing unmutated and mutant Beclin-1 in cells,autophagy levels were higher in the mutated group than in the unmutated group,apoptosis and necrosis levels were much lower in the variant group than in the unmutated group.Conclusion During the reoxygenation of rat H9c2 cardiomyocytes after hypoxia for 5hours,inhibition Caspase-3-mediated cleavage at position 147 of Beclin-1 in H9c2 increases autophagy levels and reduces apoptosis and necrosis levels...