Role of microRNAs in heart diseases miRNA induced myocardial interstitial fibrosis Baofeng Yang, Zhiguo Wang, Yanjie Lu, Zhenwei Pan Department of Pharmacology, Harbin Medical University State-Province Key Laboratories

Background In China, about 3 million people died each year of cardiovascular disease, approximately 45% of the total mortality. About 600,000 cardiogenic sudden death occurred each year, more than 90% induced by malignant arrhythmias. Myocardial interstitial fibrosis can result in structural remodeling, and induce lethal arrhythmias. Harbin Medical University

Fibrosis is a characteristic of all forms of cardiac diseases of different origins Ventricular tachycardia Myocardial fibrosis Structural remodeling Ischemia Hypertrophy Atrial fibrillation Heart failure

Mechanism of myocardial interstitial fibrosis? Electrical remodeling ? Heart diseases Structural remodeling Myocardial fibrosis

Induction of atrial fibrosis by nicotine in dogs Control Nicotine (50) Collagen content Fibrosis

Induction of atrial fibrosis by nicotine in dogs TGF-β1 TGF-βR2 CTGF Nicotine (50) Nicotine (5) Nicotine (50) Nicotine (5) Nicotine (5) Nicotine (50) Control Control Control 25kDa 70kDa 38kDa GAPDH GAPDH GAPDH

Intracardiac pacing induced fibrosis Collagen content Control Pacing Fibrosis

Intracardiac pacing induced fibrosis

Myocardial ischemia induced fibrosis Infarction area Collagen content Sham (LV) MI (Border zone) Fibrosis

Myocardial ischemia induced fibrosis NIZ IZ NIZ IZ

How fibrosis related signaling pathway was regulated ?

Alteration of expression of miRNAs in different fibrosis models Canine A-TP Canine nicotine Rat MI

Alteration of expression of miRNAs in different cardiac fibrosis patients Atrial fibrillation patients Smoking patients CAD patients

miR-133 and miR-590 regulate TGF-β1 and TGF-βRII protein expression

miR-133 and miR-590 target TGF-β1 and TGF-βRII 3’UTR miR-133 & TGF-β1 3’UTR miR-590 & TGF-βRII 3’UTR

Cardiac-specific overexpression of miR-328 causes fibrosis and cardiac dysfunction Collagen content WT TG Fibrosis

Knockdown of miR-328 protective against fibrosis induced by MI MI Border zone Infarction area WT miR-328 Knockdown WT Fibrosis miR-328 Knockdown

MiR-328 regulates collagen production

Elevated TGF-β signals in TG mice heart miR-328 M-miR-328 +AMO-328 NC TGF-β1 12KDa GAPDH 36KDa

Elevated TGF-β signals in TG mice heart P-smad2/3 miR-328 +AMO-328 M-miR-328 NC p-smad2/3 60KDa GAPDH 36KDa

MiR-328 targets directly to TGFBRIII TGF-βRIII

MiR-328 targets directly to TGFBRIII Luciferase: miR-328 targets TGFBRIII 3’UTR

miR-328 overexpression incuced fibrosis and triggered AF Canine atrium Mice ECG

miR-328 targets at CACNA1C and CACNB1

miR-328 overexpression caused cardiac fibrosis Ischemia Pacing Fibrosis miR-328 Collagen AF TGF-β1 TGFBRIII TGFBRI/II Under certain pathological conditions, level of miR-328 is increased and triggers down-regulation of its target gene TGFBR3 in cardiac fibroblast, which is leading to production of collagens and formation of cardiac fibrosis. Harbin Medical University

miRNAs and cardiac fibrosis Hypertrophy Heart Failure Myocardial Infarction Diabetic Cardiomyopathy Atrial Fibrillation miR-328 miR-133 miR-590 Calcineurin Cav1.2/ICa CACNAB1/Cavβ1 TGF-βRIII TGF-βRII TGFβ1 repolarization structure Ca2+ handling dispersion collagen Fibrosis Circulation, 2010; Cardiovasc Res, 2009a; Hypertension, 2009; BJP, 2009a, 2009b Harbin Medical University

Problems in clinical application of miRNAs Effective drug delivery way Specific action on the target tissue Selection of miRNAs carriers 纳米技术：最近美国科学家通过试验发现，可以用一种纳米微粒来包裹小分子RNA，再在其外包裹一层传递蛋白，这样就可以帮助的顺利的进入人体细胞。蛋白质外壳会在细胞内环境下自行溶解，释放出携带抗病RNA的纳米微粒。使用这种技术，能够有效的治疗白鼠感染的尤文氏肉瘤。 锁核苷酸技术：丹麦制药公司Santaris Pharma近日宣布SPC3649(LNA-antimiRTM-122)开始进入人体第一阶段的临床试验，这是世界上首个在人体中试验的microRNA药物。SPC3649是由Santaris Pharma开发的丙型肝炎的新型治疗手段， 胆固醇：针对癌基因性质的miRNA（如miR-155）合成互补的AMO，可能使致癌性miRNA失活，减缓肿瘤生长。AMO经2’-O-甲基修饰稳定性增加,毒性降低。目前正在进行小鼠体内与胆固醇结合的AMO的多器官注射实验，它成为新的miRNA抑制性治疗药物。

Advances in clinical application of miRNAs Nanotechnology：American scientists used nano-particles to wrap miRNAs, and then passes on the outer package layer of protein. This technique can effectively treat mice with the Ewing's sarcoma. Locked nucleic acids technique：Santaris Pharma, a Danish company has commenced a Phase I human volunteer trial of SPC3649 (LNA-antimiRTM-122, developed as a new approach to treat hepatitis C infection), the world's first miRNA medicine to be tested in man. Cholesterol-conjugated AMO：Complementary AMO targeted oncogene miRNA (eg miR-155) may cause carcinogenic miRNA inactivation, slow tumor growth. AMO combined with cholesterol was taken as a new treatment way to inhibit oncogene miRNA in mice. 纳米技术：最近美国科学家通过试验发现，可以用一种纳米微粒来包裹小分子RNA，再在其外包裹一层传递蛋白，这样就可以帮助的顺利的进入人体细胞。蛋白质外壳会在细胞内环境下自行溶解，释放出携带抗病RNA的纳米微粒。使用这种技术，能够有效的治疗白鼠感染的尤文氏肉瘤。 锁核苷酸技术：丹麦制药公司Santaris Pharma近日宣布SPC3649(LNA-antimiRTM-122)开始进入人体第一阶段的临床试验，这是世界上首个在人体中试验的microRNA药物。SPC3649是由Santaris Pharma 胆固醇：针对癌基因性质的miRNA（如miR-155）合成互补的AMO，可能使致癌性miRNA失活，减缓肿瘤生长。AMO经2’-O-甲基修饰稳定性增加,毒性降低。目前正在进行小鼠体内与胆固醇结合的AMO的多器官注射实验，它成为新的miRNA抑制性治疗药物。

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