Mechanism Of Glycolysis In Myocardial Cells Induced By Coxsackie Virus B3 Infection To Promote Viral Replication

Objective:To explore the effects and mechanism of glycolysis on CVB3 viral replication in myocardial cells,we detect glucose consumption and lactate production.Then we used glycolysis inhibitors to detect viral replication and autophagy in myocardial cells.Methods:（1）The cell models of CVB3-infected rat cardiomyocyte H9c2 cells and HL-1 cells were established by 5×100TCID₅₀ CVB3 virus solution.To explore changes of glucose metabolism in CVB3-infected H9c2 cell and HL-1 cell,each cell lines were divided into two groups:mock group and CVB3 group.The intervention was terminated at 24 hours post infection（hpi）.First,we detect glucose consumption and lactate production.And the total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of GLUT4,HK2,PFKM and PKM2 were detected by real-time quantitative polymerase chain reaction（RT-q PCR）.And protein levels of GLUT4,HK2,PFKM and PKM2 were detected by Western Blot.（2）To explore the effects of inhibition of HK2 and PFKM on CVB3infection in H9c2 cells,cells were divided into three groups:CVB3 group,H9c2 cell infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and 2DG;CVB3+SCT group,cells infected with CVB3virus and SCT.The intervention was terminated at 24 hours post infection（hpi）.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of CVB3 were detected by RT-q PCR.And protein levels of VP1 were detected by Western Blot.（3）To explore the effects of inhibition of HK2,PFKM and PKM2 on CVB3 infection in HL-1 cells,cells were divided into four groups:CVB3group,HL-1 cell infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and 2DG;CVB3+SCT group,cells infected with CVB3 virus and SCT;CVB3+shikonin group,cells infected with CVB3 virus and shikonin.The intervention was terminated at 24 hours post infection（hpi）.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of CVB3 were detected by RT-q PCR.And protein levels of VP1 were detected by Western Blot.（4）To examine whether glycosis had an effect on the replication of CVB3 in H9c2 cell and HL-1 cell,each cell lines were divided into two groups:mock group and CVB3 group.We used PS48 to induce a Warburg-like metabolic state,and the virus replication level was detected.（5）To explore the effects of inhibition of HK2 and PFKM on autophagy in CVB3-infected H9c2 cells,cells were divided into four groups:mock group;CVB3 group,H9c2 cell infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and 2DG;CVB3+SCT group,cells infected with CVB3 virus and SCT.The intervention was terminated at 24 hours post infection（hpi）.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of ATG5 and Beclin1were detected by RT-q PCR.And protein levels of LC3II/LC3I ratio,P62,ATG5 and Beclin1 were detected by Western Blot.（6）To explore the effects of inhibition of HK2,PFKM and PKM2 on autophagy in CVB3-infected HL-1 cells,cells were divided into five groups:mock group;CVB3 group,HL-1 cell infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and 2DG;CVB3+SCT group,cells infected with CVB3 virus and SCT;CVB3+shikonin group,cells infected with CVB3 virus and shikonin.The intervention was terminated at 24 hours post infection（hpi）.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of ATG5 and Beclin1were detected by RT-q PCR.And protein levels of LC3II/LC3I ratio,P62,ATG5 and Beclin1 were detected by Western Blot.（7）To explore the effects of autophagy on CVB3 replication,H9c2cells were divided into five groups:CVB3 group,H9c2 cell infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and2DG;CVB3+SCT group,cells infected with CVB3 virus and SCT;CVB3+2DG+rapamycin group,cells infected with CVB3 virus and 2DG and rapamycin;CVB3+SCT+rapamycin group,cells infected with CVB3virus and SCT and rapamycin.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of CVB3 were detected by real-time quantitative polymerase chain reaction（RT-q PCR）,and protein levels of VP1 were detected by Western Blot.（8）To explore the effects of autophagy on CVB3 replication,HL-1cells were divided into seven groups:CVB3 group,HL-1 cells infected with CVB3 virus;CVB3+2DG group,cells infected with CVB3 virus and2DG;CVB3+SCT group,cells infected with CVB3 virus and SCT;CVB3+shikonin group,cells infected with CVB3 virus and shikonin;CVB3+2DG+rapamycin group,cells infected with CVB3 virus and 2DG and rapamycin;CVB3+SCT+rapamycin group,cells infected with CVB3virus and SCT and rapamycin.CVB3+shikonin+rapamycin group,cells infected with CVB3 virus and shikonin and rapamycin.The intervention was terminated at 24 hours post infection（hpi）.The total RNA and total protein were extracted from cell samples at the intervention time in this study.The m RNA levels of CVB3 were detected by real-time quantitative polymerase chain reaction（RT-q PCR）,and protein levels of VP1 were detected by Western Blot.Results:（1）CVB3 infection induced increased glucose consumption and lactate production in H9c2 cell and HL-1 cell（P<0.05）.（2）The expressions of GLUT4 and key glycolysis enzymes,HK2and PFKM,were increased in CVB3-infected H9c2 cells（P<0.05）.The expressions of GLUT4 and key glycolysis enzymes,HK2,PFKM and PKM2,were increased in CVB3-infected HL-1 cells（P<0.05）.（3）At 24 hpi,2DG and SCT dramatically decreased CVB3replication in H9c2 cells without measurable effects on H9c2 cell viability and CK-MB levels.（4）At 24 hpi,2DG,SCT and shikonin dramatically decreased CVB3replication in HL-1 cells without measurable effects on HL-1 cell viability and CK-MB levels.（5）Exposure to PS48 dramatically promoted CVB3 replication in H9c2 cells and HL-1 cells.（6）At 24 hpi,compared with the CVB3 infected H9c2 cell,m RNA expressions and protein expressions of ATG5 and Beclin 1 were decreased in treatment groups of glycolysis inhibitors.And the protein expression of LC3II/LC3I ratio and P62 were decreased.（7）At 24 hpi,compared with the CVB3 infected HL-1 cell,m RNA expressions and protein expressions of ATG5 and Beclin 1 were decreased in treatment groups of glycolysis inhibitors.And the protein expression of LC3II/LC3I ratio and P62 were decreased.（8）At 24 hpi,Compared with glycolysis inhibitors treated CVB3infected cells,rapamycin significantly increased CVB3 replication.Conclusions:（1）CVB3 infection induces glycolysis in myocardial H9c2 cells and HL-1 cells.（2）Glycolysis involved in CVB3 replication in myocardial H9c2cells and HL-1 cells.（3）Glycolysis subvert the autophagy to favor CVB3 infection in H9c2 cells and HL-1 cells.