单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,Reciprocal Repression between MicroRNA-133,and Calcineurin Controls Cardiac Hypertrophy:,A Novel Mechanism for Progressive,Cardiac Hypertrophy,De-Li Dong,Yan-Jie Lu,Bao-Feng Yang,(,董德利 吕延杰 杨宝峰,),Department of Pharmacology,Aug.20-22,2021.Dalian,Background,Calcineurin/NFAT pathway induces Cardiac Hypertrophy,Calcineurin activation in cardiac hypertrophy,Decreased miR-133 expression in cardiac hypertrophy,Overexpression of miR-133 inhibits cardiac hypertrophy,Inhibition of miR-133 induces cardiac hypertrophy,Aug.20-22,2021.Dalian,1.Calcineurin-Dependent Cardiac Hypertrophy,Jeffery D.Molkentin et al.Cell,1998;93,215228.,Aug.20-22,2021.Dalian,1.Calcineurin-Dependent Cardiac Hypertrophy,Jeffery D.Molkentin et al.Cell,1998;93,215228.,Aug.20-22,2021.Dalian,1.Calcineurin-Dependent Cardiac Hypertrophy,Jeffery D.Molkentin et al.Cell,1998;93,215228.,Aug.20-22,2021.Dalian,Overexpression of calcineurin in mouse causes sudden cardiac death associated with decreased density of K channels,Deli Dong.et al.Cardiovascular Research 57(2003)320332,Aug.20-22,2021.Dalian,2.Calcineurin Activation in cardiac hypertrophy,Hae W.Lim et al.Circulation.2000;101:2431-2437.,Aug.20-22,2021.Dalian,3.Calcineurin/NFAT Coupling Participates in Pathological,but,not Physiological,Cardiac Hypertrophy,Wilkins BJ,et al.Circ Res.2004;94:110-118.,Aug.20-22,2021.Dalian,Car A et al.Nat Med.2007;13(5):613-8.,4.Decreased miR-133 expression in cardiac hypertrophy,Aug.20-22,2021.Dalian,5.Overexpression of miR-133 inhibits cardiac hypertrophy,Car A et al.Nat Med.2007;13(5):613-8.,Aug.20-22,2021.Dalian,6.Inhibition of miR-133 induces cardiac hypertrophy,Car A et al.Nat Med.2007;13(5):613-8.,Aug.20-22,2021.Dalian,Hypothesis:,Interaction between miR-133 and calcineurin in cardiac hypertrophy?,Calcineurin is the target of,miR-133,;,The expression of,miR-133,is regulated by calcineurin/NFAT signaling pathway.,Calcineurin,miR-133,Cardiac hypertrophy,Aug.20-22,2021.Dalian,Results,1.miR-133 Targets Calcineurin,Figure 1.Post-transcriptional Repression of Calcineurin by,miR-133,.(A)The sequences showing the unique sites of miRNA:mRNA complementary between,miR-133,and calcineurin for rat and mouse genes.(B)Co-transfection of,miR-133,with the luciferase reporter gene linked to the 3UTR of calcineurin reduced luciferase activity in rat and mouse species.*,P,0.01,vs,Control;#,P,0.05,vs,miR-133,.,0,0.5,1.0,Relative luciferase activity,0,0.5,1.0,NC,miR-133,+,AMO-133,miR-133,AMO-133,Mut-133,*,#,#,*,B,Mouse(n=8),Rat(n=10),5-UUGGUCCCCUUCAACCAGCUGU-3,mmu-miR-133a/rno-miR-133a,Rat,PP2B,3UTR(NM_017042),Rat,miR-133,3-UGUCGACCAACUUCCCCUGGUU-5,1600-AACCAUAGUGCCCAGUGACCAG-1621,2225-UGAACAAGAUCCAAAAAACCAA-2246,3-UGUCGACCAACUUCCCCUGGUU-5,Rat,PP2B,3UTR(NM_017042),Site 1,Site 2,Mouse,PP2B,3UTR(NM_008914),Mouse,miR-133,3-UGUCGACCAACUUCCCCUGGUU-5,1739-AGGAGCUGGAGGGGUUGACCAA-1760,A,Aug.20-22,2021.Dalian,Results:,miR-133 Targets Calcineurin,Fig.2.(A)transfection of miR-133 specifically increased miR-133 level in cultured cardiomyocytes.*P0.01 vs non-transfection group.(B)The negative control(NC)had no effect on miR-133 level,but AMO-133 decreased miR-133 level.*P0.01 vs control(lipofectamine transfection agents).n=3.,A,B,Aug.20-22,2021.Dalian,Results:,miR-133 Targets Calcineurin,Fig.3.Effects of,miR-133,on calcineurin protein levels(n=5),mRNA level(n=5)and cell area(The number was in brackets)in normal cultured cardiomyoctes.*,P,0.01 vs Control.,Aug.20-22,2021.Dalian,A,B,C,1.miR-133 Targets Calcineurin,Figure 4.Effects of,miR-133,on calcineurin protein levels(n=5),mRNA level(n=5)and cell area(The number of cells was in brackets)in cardiomyoctes treated with PE(50M).,A,B,C,Aug.20-22,2021.Dalian,Results:,Calcineurin/NFAT Signaling Regulates,miR-133,Expression,Fig.5.Calcineurin inhibitor CsA inhibited cardiac hypertrophy induced by transverse aortic constriction in mice.,Sham,TAC,TAC+CsA,Aug.20-22,2021.Dalian,A,B,Results:,Calcineurin/NFAT Signaling Regulates,miR-133,Expression,Fig.6.Calcineurin inhibitor CsA inhibited cardiac hypertrophy(The number was in brackets),calcineurin expression(n=5)and activity(n=5).*,P,0.01,vs,Control;#,P,0.01,vs,TAC.,Aug.20-22,2021.Dalian,A,B,C,Results:,Calcineurin/NFAT Signaling Regulates,miR-133,Expression,Fig.7.CsA restored the decreased,miR-133,expression but not the increased,miR-195,expression in TAC-induced hypertrophic heart.*,P,0.01 vs Control.#,P,0.01 vs TAC;n=5,.,Aug.20-22,2021.Dalian,A,B,Results:,Calcineurin/NFAT Signaling Regulates,miR-133,Expression,Fig.8.Primary rats cardiomyocytes in serum-free medium were stimulated with PE and PE+CsA.Cardiomyocytes were identified with a-actinin antibody(red signal)and nuclei were stained with bis-benzamide(blue).,Control,PE,PE+CsA,Aug.20-22,2021.Dalian,A,B,Results:,Calcineurin/NFAT Signaling Regulates,miR-133,Expression,Fig.9.CsA inhibited hypertrophy(The number was in brackets),calcineurin expression(n=5)and activity(n=5)in cardiomyocytes treated with PE(50M).*,P,0.01,v