1. Role of microRNAs in Atrial Fibrillation Baofeng Yang Yanjie Lu Department of Pharmacology, State-Province Key Laboratories, Harbin Medical University

2. 2 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●  Atrial fibrillation (AF), the most common cardiac arrhythmia.  AF increases with age, occurs in 8% of people over 80, and causes a third of all strokes.  AF can cause or exacerbate heart failure. Harbin Medical University Background

3. 3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Sinus rhythmAtrial fibrillation

4. 4 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Positive feedback-loops of atrial remodeling in AF Cardiovascular Research 54 (2002) 230–246 AF begets AF

5. 5 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Involvement of microRNAs in heart  Myocyte growth, integrity of the ventricular wall, contractility, angiogenesis, gene expression, maintenance of cardiac rhythm.  In multiple forms of heart disease, hypertrophy, fibrosis, arrhythmia etc.  microRNAs (miRNAs) as biomarker for cardiovascular disease.

6. 6 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miRNAs and cardiac fibrosis Journal of Cardiovascular Pharmacology. 2010, 454-459

7. 7 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Nicotine increases AF vulnerability in dogs Cardiovascular Research (2009) 83, 465–472

8. 8 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Induction of atrial fibrosis by nicotine in dogs ControlNicotine (50) Fibrosis Collagen content Cardiovascular Research (2009) 83, 465–472

9. 9 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Effects of nicotine on expression of TGF-b1 and TGF-bRII in cultured canine atrial fibroblasts AB Cardiovascular Research (2009) 83, 465–472

10. 10 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Nicotine-induced alteration of expression of miRNAs in cultured atrial fibroblasts and atrial tissue Cardiovascular Research (2009) 83, 465–472

11. 11 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-133 and miR-590 regulate TGF-β1 and TGF-βRII protein expression TGF-β1TGF-βRII

12. 12 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Effect of miR-133 and miR-590 on collagen production in cultured canine atrial fibroblasts

13. 13 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Intracardiac pacing induced fibrosis Control Fibrosis Pacing

14. 14 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Myocardial ischemia induced fibrosis Sham (LV)MI (Border zone)

15. 15 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Alteration of miRNAs profiling in A-TP dogs Canine A-TP

16. 16 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Alteration of expression of miRNAs in AF patients Atrial fibrillation patients

17. 17 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets directly to TGFBRIII

18. 18 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets directly to TGFBRIII Luciferase: miR-328 targets TGFBRIII 3’UTR

19. 19 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets directly to TGFBRIII

20. 20 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● p-smad2/3 GAPDH 36KDa NC miR-328 +AMO-328 M-miR-328 60KDa Elevated TGF-β signals by miR-328

21. 21 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 regulates collagen production in fibolasts

22. 22 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Knockdown of miR-328 protective against fibrosis induced by MI antagomiR-328 MI MI Border zone MI A-328

23. 23 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Collagen TGFBRI/II TGFBRIII Pacing miR-328 Fibrosis TGF-β1 AF Harbin Medical University Signaling of miR-328-induced cardiac fibrosis Ischemia

24. 24 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miRNA and ion channels

25. 25 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Regulation of channels by miRNAs miR-1 Kir2.1 (KCNJ2)Cx43 (GJA1) miR-26 ICaL (CACNA1C CACNB1) miR-328 IKr (KCNH2) miR-133

26. 26 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Alteration of electrophysiology in A-TP dogs

27. 27 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miRNA expression profiling in atrial samples from a canine model of AF and AF patients

28. 28 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 overexpression triggered AF Canine atrium Mice ECG

29. 29 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Alignment of the sequences of miR-328 with the 3‘-UTRs of CACNCA1 and CACNB1

30. 30 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets at CACNA1C and CACNB1

31. 31 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 affected I Ca-L currents A B miR-328 TG mice

32. 32 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets at CACNA1C and CACNB1

33. 33 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 targets at CACNA1C and CACNB1 AB

34. 34 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miR-328 did not affect IK1 and Ito A B Circulation 2010 Dec 7;122(23):2378-87

35. 35 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● miRNA Perspective  Biomarkers in disease diagnosis and progression  Potential therapeutic targets by blocking or mimicking miRNA activities

36. 36 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

37. 37 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Harbin Medical University Team members Baofeng Yang Yanjie Lu Xu Gao Hongli Shan Jing Ai Baoxin Li Guofen Qiao Chaoqian Xu Wenfeng Chu Benzhi Cai Yong Zhang Deli Dong Zhenwei Pan Jiening Xiao Huixian Lin Ning Wang Lihua Sun Yan Liu Zhiguo Wang Yunlong Bai Dongfang Gu Ying Zhang

38. 38 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 1. APD:action potential duration; AFCl: AF cycle length; θ: conduction velocity; WL: wave length. 2. in plasma samples isolated from patients with acute myocardial infarction, cardiac myocyte-associated mature miR-1, miR-208b and -499 were greatly elevated when compared with controls 3. These findings suggest that miR-24 has a critical role in cardiac fibroblast function and cardiac fibrosis after MI through a furin–TGF-β pathway. MicroRNA-24 Regulates Cardiac Fibrosis after Myocardial Infarction JCMM. 1582-4934.2012.

39. 39 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● FIGURE 1. miRNAs implicated in cardiac fibrosis. Different injuries to the heart induce hypertrophy and fibrosis at least in part by microRNA-mediated mechanisms. Upregulation of miR-21 in fibroblasts downregulates Spry1, which derepresses ERK/MAP kinase signaling and results in enhanced fibroblast survival and extracellular matrix deposition. Downregulation of PTEN by miR-21 in fibroblasts may increase matrix metalloproteinase-2 expression. Downregulation of miR-29 in fibroblast may simulate fibrosis by enhanced collagen and fibrillin expression, whereas miR-30 downregulation derepresses CTGF expression, a powerful stimulator of cardiac fibrosis. CTGF expression is also increased in cardiomyocytes after downregulation of miR-133. miR-590 downregulation in cardiomyocytes increases TGF beta expression, which is also linked to fibrosis by stimulating CTGF expression by downregulation of miR-30 and miR-133 expression. Secretion of FGF-2 by fibroblasts may stimulate cardiomyocyte hypertrophy.

40. 40 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 5 and 50 nmol/L kg (i.v.) nicotine yielded blood levels of 50 and 500 nmol/L, respectively, at 1 h after administration (administrated for 30 days). It has been shown that the average blood concentration of nicotine in regular smokers is 220 nmol/L and that the level can reach 440 nmol/L after consumption of a single cigarette Mecamylamine (MA, a centrally acting antagonist selective for non-a7-nAChRs, a-Bungarotoxin (a-BTX, an antagonist selective to a7 nicotinic acetylcholine receptors, a7-nAChRs, Hexamethonium (HEX, a non-competitive antagonist to peripherally acting or ganglionic nAChR), SB-431542, a small molecule inhibitor of TGF-bRI

41. 41 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● How fibrosis related signaling pathway was regulated by miRNA?

42. 42 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Model showing positive regulation of immediate-early TGF-ß signaling resulting in the induction of ECM. LTBP/LAP = latent TGF-ß binding protein; SBE = Smad binding element; TGF-ßR = TGF-ß receptor; TFBE = transcription factor binding element, whose identity depends on the identity of the TGF-ß-responsive promoter, P = phosphorylated protein. LTBP/LAPs are cleaved from latent TGF-ß, releasing active TGF-ß. TGF-ß then binds to TGF-ßR I and II. TGF-ßRI phosphorylates Smad3, which binds to Smad4. The resultant complex migrates into the nucleus to interact with the SBE and basal transcription factors binding to the TFBE. Activity of the Smad/basal transcription factor complex is modified by other signaling cascades depending on the promoter of interest. The net result is the induction of gene expression, including the elevated expression of genes encoding extracellular matrix. For details, see text._art;1>

43. 43 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●