Montréal (Québec) Canada H2W 1R7 cyclic GMP: lessons from ... · Montréal (Québec) Canada. H2W...

Institut de recherches cliniques de Montréal (IRCM)110, avenue des Pins ouestMontréal (Québec) CanadaH2W 1R7

September 25, 200963rd High Blood Pressure Conference 2009Lewis K. Dahl Memorial Lecture

Cardioprotective effects of cyclic GMP: lessons from animal genetic models

Affiliated to Université de Montréal

No relationship to disclose

Lewis K. Dahl: Web visibility…


Lewis K. Dahl: enduring contributions


Comparison of hypertension among geographically distinct populations with different salt intake levels• review published in:

Cottier P, Bock KD: Essential Hypertension—an InternationalSymposium. Berlin: Springer, 1960, pp. 53–65

• reprinted in: Int. J. Epidemiol. 34: 967–972 (2005)

Demonstration of the role of genetic factors in susceptibility to experimental hypertension due to chronic excess salt

• Nature 194: 480-482 (1962)• J. Exp. Med. 115: 1173-1190 (1962)• discussed in recent editorial: J. Exp. Med. 204: 1507 (2007)

Selective breeding program to establish genetic experimental models of salt-sensitive hypertension

• New rat strains: Dahl-S and Dahl-R• further established into inbred rat strains by J.R. Rapp• used in numerous animal genetic studies

Novel inbred rat strains: WKHT and WKHA


XWKY SHR

F1

F2

Syst. BP: ≡

WKY

Activity: > WKY, ≡

SHR

HW/BW: > WKY, < SHR

Phenotype selection followed by inbreeding

WKHA WKHT

Cross performed by E.D. Hendley (U. of Vermont)

Syst. BP: > WKY, ≡

SHR

Activity: ≡

WKY, < SHR

HW/BW: > WKY, < SHR

Note: WKHA are not “hyperactive”


WKY

SHR

WKHA

LEWISWISTAR

0

200

400

600

800

1000

rat strain

Loco

mot

or a

ctiv

ity

⇒ one should be cautious not to define quantitative phenotypes as “normal” or “abnormal” in absolute terms, but only in comparison to other phenotypes within a species

WKHA have concentric LVH vs. WKY


ANF in ventricles

Ventricular weight0

50

100

150

200

250

fmol

e / m

g w

et w

eigh

t

2.90

3.10

3.30

3.50

Ven

tr. w

eigh

t / b

ody

wei

ght

WKY WKHAAdditional observation: LV concentration of ANF correlates negatively with LV weight → → → →

F2 intercross between WKY and WKHA


F2

F1 X X

P1 P2n

phenotype

n

phenotype

X

n

n

phenotype

phenotype

F2 GENETIC INTERCROSS

* *

*

*** variance = environment** variance = environment x genetics

F2

F1 X XF1 X X

P1 P2n

phenotype

n

phenotype

X

n

n

phenotype

phenotype

F2 GENETIC INTERCROSS

* *

*

*** variance = environment** variance = environment x genetics

Co-segregation analysis in F2 population


L.V. weight / body weight tertile

lower mid higherlower mid higher40

60

80

100

120

fmol

e / m

g w

et w

eigh

t

ANF peptide

(31)

(72)

(41)

lower mid higher0.70

0.95

1.20

O.D

. rat

io

β-MHC mRNA

(30)

(23)

(24)

0.20

0.45

0.70

O.D

. rat

io

ANF mRNA

(18)

(45)

(25)

Circ. Res., 84:1453-1458 (1999)

Detection of QTLs for LVM and LV ANF


0 20 40 60 80 100 1200

1

2

3

4

5

LOD

(L.V

. mas

s)

0 20 40 60 80 100 1200

5

10

15

LOD

[ln(

vent

r. A

NF)

]

Mgh1 Rat126 Mgh3 Rat28 Rat173 Mgh8 Rat99Wox10 Rat245

Rat180

1-LOD interv.

1-LOD interv.

QTL/ANFv

QTL/LVM

a

b

Circ. Res. 88:222-227, 2001

→ One of the first examples of an “expression QTL in cis” (cis-eQTL) co-mapping with a whole organ QTL

Additional finding: SNP in Nppa promoter alters transcriptional activity

Physical confirmation of QTL in congenic rats


Physiol Genomics 12:61-67, 2002

←

“Cm24”

Natriuretic peptide signaling


Heart-specific guanylate cyclase transgene


Not I Xho I(Start) (Stop) (A)n

α-MHC prom CAT - GC Rabbit -globinβ

brain

heart

kidne

y

liver

muscle

lungsto

mach

splee

n

18s

18s

Str. 09

Str. 19

GC cGMP0

200

400

600

800

fmol

/ mg

prot

NT

TG*

*

J. Biol. Chem. 278:47694, 2003

Not I Xho I(Start) (Stop) (A)n

α-MHC promα-MHC prom CAT - GC Rabbit -globinβRabbit -globinβ

brain

heart

kidne

y

liver

muscle

lungsto

mach

splee

n

18s

18s

Str. 09

Str. 19

GC cGMP0

200

400

600

800

fmol

/ mg

prot

NT

TG*

*

J. Biol. Chem. 278:47694, 2003

The cardiac GC transgene blocks the hypertrophic effects of aortic constriction on:


NT TG4

5

6

7

ratio

HW/BW

NT TG50

75

100

125

mm

x 1

0-2

IVSW

NT TG50

75

100

125

mm

x 1

0-2

LVPW

**

SHAM AAC

NT TG15

20

25

30

µm

cardiomyocyte width

NT TG1000

2000

3000

4000

5000

6000

µm 2

cardiomyocyte surface

***

******

**

SHAM AAC

LV weight →

LV wall thickness →

Surface area of isolated cardiomyocytes →

J. Biol. Chem. 278:47694, 2003

The QTL Cm24 has no effect on LVM of female rats under basal conditions….


Physiol Genomics 12:61-67, 2002

…but Cm24 increases the effect of volume overload on adverse LV remodeling in female rats


Hypertension, 47:128-133, 2006

Sex-specificity of QTLs linked to LVM


Mamm. Genome 16: 700-711 (2005)

Where do we go from here?


Two directions…


• cardioprotection• mechanisms of action• therapeutic potential

Genetic markers of cardiovascular risk / outcome

o

Increasing cardiac cGMP by pharmacologic means prevents cardiac hypertrophy


Chronic inhibition of cyclic GMP phosphodiesterase 5A (by Viagra) prevents and reverses cardiac hypertrophy.

Takimoto E et al. Nat Med 11: 214 - 22 (2005)

The cardiac GC transgene prevents functional deterioration in dystrophic hearts


mdx

mdx/GC+/0

PNAS 105:7028-7033 (2008)

The cardiac GC transgene and sildenafil have similar cardioprotective effects


PNAS 105:7028-7033 (2008)

Perfused working heart preperations with acute increase in cardiac mechanical stress by isoproterenol infusion

Cardioprotection via action on mitochondria?


From: Das A. et al. Am. J. Physiol. 296: H1236 (2009)

http://ajpheart.physiology.org/content/vol296/issue5/images/large/zh40050988020009.jpeg

Sildenafil and MPTP opening (1)


[3H]‐DOG

PTP closed

PTP open

[3H]‐DOG‐6P

cardiomyocyte

(Burelle & al., unpublished

data)

Mitochondrial permeability

transition

pore opening

(MPTP)

RPP(mm Hg x bp

m)

Time(min)

Control

mdx

1 µM Norepinephrine

(Marcil et al. JMCC 2006)

LFI ReperfusionNormoxia

[3H]‐DOG

*mdx

+ Sil

*

Sildenafil and MPTP opening (2)


(Burelle & al., unpublished

data)

Index of membrane integrityLD

H re

leas

e (m

U*g

ww

*RPP

-1) * *

PTP opening

index

mdx

+ SilCtrl mdx

* *

([3 H]‐DOG ratio)

mdx

+ SilCtrl mdx

Two directions…


• cardioprotection• mechanisms of action• therapeutic potential

Genetic markers of cardiovascular risk / outcome

Phenotypic variation in young healthy adults


50 60 70 80 90 100

110

120

130

140

0

5

10

15

LVM/BSA

Perc

enta

ge

0

500

1000

1500

2000

2500

3000

0

10

20

30

Plasma NT-ANP

perc

enta

ge(- 1 SD)

66.4 ± 7.6(+ 1 SD) 119 ± 8

(- 1 SD) 224 ± 49

(- 1 SD) 2220 ± 300

Institut de recherches cliniques de Montréalhttp://www.ircm.qc.ca

Acknowledgements Students and fellows:

• Sandra Masciotra• Isabelle Boutin-Ganache• Ahmad Zahabi• Emmanuelle Souzeau• Bastien Llamas

• Swapnil Sinha• Marie-Pier Scott-Boyer

Institut de recherches cliniques de Montréalhttp://www.ircm.qc.ca

Acknowledgements Collaborators:• U. of Burlington (Vt):

Edith Hendley

• U. of Montreal: Christine Des Rosiers Yan Burelle

• McGill University: Basil Petrov Jamie Engert

• Laval University: Éric Larose Philippe Pibarot

Funding: CIHR / NHLBI

VANCOUVER HYPERTENSIONSeptember 26-30, 2010VANCOUVER HYPERTENSION 2010September 26-30 2010

www.vancouverhypertension2010.com

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