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田小利田小利人类群体遗传室人类群体遗传室北京大学分子医学研究所北京大学分子医学研究所

死亡

代偿期 失代偿期 恶化期

结构和功能的改变 --- 重塑

高血压，冠心病遗传，心肌病瓣膜病，先心病

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心力衰竭

心力衰竭的诱因和病理过程心力衰竭的诱因和病理过程

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激素、药物、

神经递质膜受体

第二信使 Ca2+

离子通道

活性肽

心力衰竭

心律失常

线粒体活性氧心脏病相关基因

活性肽

(1)

(1) 心

力

衰

竭

心力衰竭可能的发展基础心力衰竭可能的发展基础

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心力衰竭

临床诊断遗传学通道电生理学神经和信号转导

心肌重塑的关键分子

细胞水平鉴定动物模型计算机数字化模拟3-D结构研究

1. 作用机制及早期干预2. 新药靶点及可药性3. 早期诊断标记的可能性

目前主要研究的内容目前主要研究的内容

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AOGEN

Renin

ACE

ANG I

ANG II 5

Salt/water balance

Contraction of VSMC

Mitogenic effects

Neurotransmitter

Hormone-stimulation factor

Cardiac output6

PLD PLC

AII

PC

PA

DAGCholine IP3

PIP2

PKC

Mek

Mapk Stats

ER

Ca++

Shc

Jaks

SIE

C-fosC-Jun

Stats

SOSGrb2

Ras

Raf

GTP

C-mycEgr

Ca++

Ca++ Ca++

Ca++

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AOGEN

Renin

ACE

ANG I

ANG II

Kallikrein

Tonin

ChymotrypsinCapthepsin G

ChymaseKallikrein

ChymotrypsinCapthesin G

8

9

50

40

30

20

10

0

Captopril

Chymosta

tin

Cap + C

hymo

Cap + A

prot

CV 11974

%p

he

nyl

ep

hri

ne

ANG1 perfusion isolated aorta

Nishimura, 1998

Chymase:

Ang II forming enzyme in vitro

Found, cloned and characterized by Dr. Hidenori Urata

Urata, 1990 ACE

Chymase

Fe Ad

Tian, 1996

Renin

Angiotensinogen

ACE

Angiotensin I

Angiotensin II

AT1 AT2

Angiotensinogen

Renin

ACE

AT1

AT2

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Angiotensinogen

Renin

Angiotensin 1

Angiotensin 2

Kininogen

Kallikrein

Bradykinin

Inactive peptides

ACE

RAS(AII/AT1)

AS(a1)

ETS(ET-1/ETA)

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12

Angiotensin 1

Bradykinin

AcSDKP(N-acetyl-Ser-Asp-Lys-Pro)

Angiotensin IICell growth. etc.

degraded fragments

degraded fragmentsActiate TGF-β signaling

13 Fleming I et al. Physiology. 2005;20:91-5.

ACE

NH2

ACE inhibitorExtracellular

CytosolCOOH

Nucleus

Gene expression(ACE, COX-2)

CK2JNK

MKK7

P JNK cJun

cJunP

cJunP

cJunPcJun

P

AP-1cJun

PcJunP

Clinical significance of this pathway is under investigation

Angiotensinogen (M235T)

Together with ACE-D, associated male LVHCAD

AT1 (A1166C)

Associated male LVH

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Somatic promoter Germinal promoter

250 - 285 bp

I/I

I/D

D/D

Plasma Cardiac

+ +

++ ++

+++ +++

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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ACE (I/D)

Earlier than 1994: tends to associatedLater than 1994: tends to not associatedLVH of man, not womanDegree, not frequency of LVHACEI treatmentQT dispersion, heart rate variabilityExercise-induced LHV

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AortaLung

Kidney

Brain

LiverL. V

entric

le

R. ventric

le

Sk. muscl

e

rACE

hACE

In crea sed fo ld

0

1 0

2 0

3 0

4 0

5 0

6 0

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SDTGR

5060708090

100110

-10 -9 -8,5 -8 -7,5

5060708090

100110

-10 -9 -8,5 -8 -7,5

SDTGR

302520151050 SD TGR

AII (pg/g)

AI on coronary artery flowCardiac AII18

ACEI

1,61,82,02,22,42,62,83,0

LV/B

SD-sh

SD-abTGR-ab

TGR-sh

LV/B

(mg/

g)

012345678

ANF Col3

%G

AP

DH

0

20

40

60

80

100

120

140

160

180

SD-sh

TGR-sh

SD-ab

TGR-ab

SD-shTGR-shSD

-ab

TGR-ab

ANF

Col3

GAPDH

SD-sh

SD-abTGR-ab

TGR-sh

SBP

(mm

Hg)

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20

Age > 10 months

col1

col3

GAPDH

NTGRTGRL1172

TGRL1173

Lisinopril

AcSDKP

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ACE

TGF-β

TGF-R1

Smad3Smad2

Smad4

Col

TGF-β

GAPDH

Smad3-p

Smad3

Smad2-p

Smad2

AcSDKP

NTGRTGRL1172

TGRL1173

NTGRTGRL1172

TGRL1173

Lisinopril

AcSDKP

NTGRTGRL1172

TGRL1173

Lisinopril

AcSDKP

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Transcriptional machinery

CmGH-actEpigenetic modification

Definition of CG island in RAS Definition of CG island in RAS promoterspromoters

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ATG

TSS

5-UTR

1 kb

CG sites

CTGTCCCTGGCTCCTTCCCTGATCCCACCGCCAGCCTCACCCCACGGTTCCTCCATTGCCCCACCTCCCACTGCGCCGCCGGGCCTCTGCCAGGGTCAAGGGGCTTCCCCCCTCTGGCAGCAGACGCCATGGTGCCGAGGTGGCCTCCACAACCGCCCTGTGCGCCAATAGGACAAGACTGTCCTCCCTCCCCCACACTTGTCACTTTGAGGGACACGTGGATGAGACAGGAAAACACAGGGGAGTGTGGAGACCTGAGGTGACTTGGAGCAAGCCTCTCAACCTGAGCGGCAATTTCTTCATCTGTAAAATGAGGGGGTTGTTCTCATCTCTGAGGCTTTGTGTCGCTCTCAAAGCCTGCTAGCCTCGGGTTCTAGGACTCTGTTGGGATCGTGTGTGATGTTTTCTGCTGAGCGACGGCAGCCTGTGTCCTCGGGGGGAAAGAGGGCAGGCGCTCCAAAGCTCCTGCGCTCTGTGGCTCCCCCTCCCTCGCAGCCCCAAGCCCCAGGTGTGCCGGCCGCCCTGAGCCCCTCCAGCACCTCCCGGAGGCGCCTGCAAGACACCTAAGGTCCCCGCCTCCCTCCTCTCCCCCCCGCCACACCCCTACCCCCGGCAGGCGACGTCCCCGCCCCTCGACCATGGCCTGGTGAAGAAGCCGGCCAGGCCCGATCAGCCCCATCCCCGCCGCACGAGCGGCGCCTGCGGACAGCTCCTGGGGCCCCGGCCTTGTCACTCCGGAGGCGGGAGGCTCCGGGGGGTCGGGCTGGGAAGATCGAGCCGGAGGCCGCTAGGCTCCCAGGCCCCGGCCGAGGCTGCGCGGCCGCACGGTGGGCAGGCTCGGGTGTTCCGGCAAACTGCCGGGTCCCCATCTTCAAAAGAGAGGAGGCCCTTTCTCCAGCTTCCTCTGCGGGAGCCCGACCCAGCCCCATCCCGCCACCCCCGGGCTGCACCTCGGCCCCTCCCCGGCCCGCGCCCCTGCCCGGGGCGGGCCAGGAACCTCGGCCCGCGCCGCTGGGGACTTTGGAGCGGAGGAGGAAGCGCGGCGGGGCGGGGGCGGGGGTGTGTCGGGTTTTATAACCCGCAGGGCGGCCGCGGCGCAGGAGAAGGGGCAGAGCCGAGCACCGCGCACCGCGTCATGGG

ACEACE启动子区启动子区 CGCG 岛分析岛分析

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AngiotensinogenReninACEAT1aAT2

7469874588

甲基化分析的原理甲基化分析的原理

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mCG

亚硫酸氢钠

mCG PCR CG

CG UG TG

CN UN TN

克隆测序

直接测序

Characteristics of ratsCharacteristics of rats

SHR/SP （ N=20 ） WKY （ N=20 ） P value

HR （ bpm ） 376.8±34.6 306.7±29.5 0.0000

SBP(mmHg) 207.1±15.0 130.6±10.3 0.0000

DBP(mmHg) 164.8±14.9 95.2±11.9 0.0000

H/B(mg/g) 4.16±0.01 3.69±0.016 0.0000

V/B((mg/g) 3.86±0.08 3.34±0.017 0.0000

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C CG

29100% protected = 100% methylation

How to quantify methylationHow to quantify methylation

C

WKYSHR

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Methylation of AOGMethylation of AOG

CGCCCGCC

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GCCCGCCC

WKYSHR

Methylation of ACE Methylation of ACE (reversal (reversal sequence)sequence)

W1 S1 W2 S2 W4 S4 W12 S12

AOGEN

GAPDH

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W1 S1 W2 S2 W4 S4

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L.V 1w 2ws 4ws

AOGEN

AT1b ND ND ND

AT2 ND ND ND

Renin

ACE

AT1a ND ND ND

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Met

---

?

?

?

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Several signaling pathways are involved in the pathogenesis of heart failure

RAS plays a role in cardiac dysfunction through multiple pathways

Expression and epigenetic modifications of RAS plays a critical roles in heart failure

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谢谢谢谢