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1、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 Harbin Medical University, P.R.China,Aug.20-22,2009.Dalian,Background,Calcineu
2、rin/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.20-22,2009.Dalian,1. Calcineurin-Dependent Cardi
3、ac Hypertrophy,Jeffery D. Molkentin et al. Cell,1998; 93,215228.,Aug.20-22,2009.Dalian,1. Calcineurin-Dependent Cardiac Hypertrophy,Jeffery D. Molkentin et al. Cell,1998; 93,215228.,Aug.20-22,2009.Dalian,1.Calcineurin-Dependent Cardiac Hypertrophy,Jeffery D. Molkentin et al. Cell,1998; 93,215228.,Au
4、g.20-22,2009.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.20-22,2009.Dalian,2. Calcineurin Activation in cardiac hypertrophy,Hae W. Lim et al.Circulation. 2000;
5、101:2431-2437.,Aug.20-22,2009.Dalian,3. Calcineurin/NFAT Coupling Participates in Pathological, but not Physiological, Cardiac Hypertrophy,Wilkins BJ, et al. Circ Res. 2004;94:110-118.,Aug.20-22,2009.Dalian,Car A et al. Nat Med. 2007;13(5):613-8.,4. Decreased miR-133 expression in cardiac hypertroph
6、y,Aug.20-22,2009.Dalian,5. Overexpression of miR-133 inhibits cardiac hypertrophy,Car A et al. Nat Med. 2007;13(5):613-8.,Aug.20-22,2009.Dalian,6. Inhibition of miR-133 induces cardiac hypertrophy,Car A et al. Nat Med. 2007;13(5):613-8.,Aug.20-22,2009.Dalian,Hypothesis: Interaction between miR-133 a
7、nd 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.20-22,2009.Dalian,Results 1. miR-133 Targets Calcineurin,Figure 1. Post-transcriptional Repression o
8、f 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 sp
9、ecies. *P0.01 vs Control; # P0.05 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),A,Aug.20-22,2009.Dalian,Results: miR-133 Targets Calcineurin,Fig.2. (A) transfection of miR-133 specifically increased miR-133 l
10、evel 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.20-22,2009.Dalian,Results: miR-133 Targets Calcineurin,Fig.3. Effe
11、cts 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. *P0.01 vs Control.,Aug.20-22,2009.Dalian,A,B,C,1. miR-133 Targets Calcineurin,Figure 4. Effects of miR-133 on calcineurin protein levels (n=5), mRNA level
12、 (n=5) and cell area (The number of cells was in brackets) in cardiomyoctes treated with PE (50M).,A,B,C,Aug.20-22,2009.Dalian,Results: Calcineurin/NFAT Signaling Regulates miR-133 Expression,Fig.5. Calcineurin inhibitor CsA inhibited cardiac hypertrophy induced by transverse aortic constriction in
13、mice.,Sham,TAC,TAC+CsA,Aug.20-22,2009.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). *P0.01 vs Control; #P0.01 vs TAC.,Aug.20-22,
14、2009.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. *P0.01 vs Control. # P0.01 vs TAC; n=5.,Aug.20-22,2009.Dalian,A,B,Results: Calcineurin/N
15、FAT 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.20-22,2009.Dalian,A,B,Re
16、sults: 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). *P0.01 vs Control. # P0.01 vs PE.,Aug.20-22,2009.Dalian,A,B,C,Results: Calcineurin/NF
17、AT Signaling Regulates miR-133 Expression,Fig.10. CsA alleviated the decreased miR-133 expression in PE-treated cardiomyocyes. *P0.01 vs Control. #P0.05 vs PE; n=5.,Aug.20-22,2009.Dalian,Results: Calcineurin/NFAT Signaling Regulates miR-133 Expression,Fig.11. Calcineurin/NFAT-dependent Regulation of
18、 miR-133. (A) Antisense-A ODNs increased the expression of miR-133 in cultured cardiomyocytes. *P0.01 vs Control; n=4. (B) NFAT decoy ODNs increased the expression of miR-133 in cultured cardiomyocytes. *P0.01 vs Control; n=5.,Aug.20-22,2009.Dalian,Conclusion:,Aug.20-22,2009.Dalian,Chang Chen, Rong Huo, Ning Wang, Zhe Li, Yu-Jie Tu,Acknowledgments,Jun-Tao Hu, Xia Chu, Wei Huang, Yan-Jie Lu Bao-Feng Yang,Aug.20-22,2009.Dalian,Thank you,