Modificazioni età-correlate del signaling β-adrenergico e basi molecolari dell'utilizzo

dei β-bloccanti

Dario LeoscoCattedra di GeriatriaUniversità degli Studi di Napoli “Federico II”

53° Congresso NazionaleSocietà Italiana di Gerontologia e Geriatria

Firenze, 26-29 Novembre 2008

“L’Italia? Non è un paese per vecchi”

CARDIOMYOCYTE

extracellular

intracellularACβ-AR

CACA CACA

CACA

CACACACA

CACA CACA

CACACACA

CACA

CA

ATP cAMP

Gsαβγ

PKA+

contractility

Physiologic conditions

CA

Gsα

CARDIOMYOCYTE

extracellular

intracellularACβ-AR

Gsαβγ

PKA

ATP cAMP

Heart Failure

CACA CACA

CACA

CACACACA

CACA CACA

CACACACA

CACACACA

CACA CACA

CACA

CACACACA

CACA CACA

CACACACA

CACA

CACA CACA

CACA

CACACACA

CACA CACA

CACACACA

CACA

CACACACA

PI3KPI3K

P

GRK2

↓contractility

Heart Failure

CACA CACA

CACA

CACACACA

CACA CACACACA

CACACACACACA

CACA CACA

CACA

CACACACA

CACA CACACACA

CACACACA

CACA CACA

CACA

CACA

CACACACA CACA

CACACACA

CACA

CACACACA

P

GRK2

CACACACA

β-AR

Cardiomyocyte

Extracellular

Cytosol GRK2 inducesβ-AR INTERNALIZATION

β-AR downregulation and desensitization occur

also with“Aging”

Age-associated reductions of cardiac myocyteβ1AR and β2AR adrenergic responses in rats

NE concentration (M)

Con

trac

tion

Am

plitu

de(%

C)

Zinterol concentration (M)

Con

trac

tion

Am

plitu

de(%

C)

Xiao RP, Lakatta EG, Koch WJ et al. J Clin Invest 1998

0

5

10

15

20

25

30

35 β1ARβ2AR

2 mo 24 mofm

ol/m

g m

embr

ane

prot

ein p< 0.01

β-AR function changes with age of subject in myocytes from non failing human ventricle

Davies CH, Ferrara N, Harding SE. Cardiovasc Res, 1996

β-Adrenergic Receptor desensitizationThe role of GRK2 (β-ARK1)

The β-Adrenergic Receptor Kinase(βARK1 or GRK2)

• Ubiquitously expressed member of the GRK family.

• Highest expressed GRK in the heart.• Up-regulated (heart and blood vessels) in

several cardiovascular disorders including heart failure and hypertension.

• Basic science research allowed for a way to inhibit the activity of this enzyme (preventing membrane translocation -βARKct).

β1-AR blockade enhances contractility reserve of the aged heart via restoring cardiac β-AR signaling

†

Old + β1AR blockadeOld + Exercise

Old

Young

fmol

/mg

β-ARs density

0

10

20

30

40

50

60

70

*

pmol

/mg/

min

cAMP production

0

10

20

30

40

50

60

70

* *Old Young Old+β1 block Old Young Old+β1 block

Old YoungGRK2Actin

Old+β1 Block

Leosco D et al. Am J Physiol 2007

β-AR blockade reduces cardiac GRK2 (β-ARK1)levels in normal hearts

Iaccarino G, Lefkowitz RJ and Walter J. Koch. Circulation 1998

Harding et al. PNAS 2001

Targeting GRK2 (β-ARK1) dramatically increasessurvival in animal models of heart failure

LV Ejection Fraction

LV prior to infection LV post-infectionCardiac GRK2

Giuseppe Rengo and Walter J. Koch. Circulation 2008

Cardiac GRK2 normalization via β1AR blockadeand AAV6-βARKct Gene Therapy

Improves Cardiac Function in Heart Failure

Cardiac GRK2 normalization via β1AR blockadeand AAV6-βARKct gene therapy

normalizes the neurohormonal axis in heart failure

Giuseppe Rengo and Walter J. Koch. Circulation 2008

Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007

Inhibition of adrenal GRK2 activity leads to reduction of circulating cathecolamines

Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007

Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007

Inhibition of adrenal GRK2 activity leads to improved function of the failing heart.

Pathophysiologic role of GRK2 and the therapeuticpotential of its inhibition in heart failure.

Novel pathophysiologic implications of β-blocker therapy in heart failure

• Potential action of β-blockers of third generation (carvedilol, nebivolol) on regulating cathecolaminessecretion from the adrenal gland

Kajiwara K et al. J Pharmacol Exp Ther 2002

GRK2 as a Novel Biomarkerof Heart Failure in Humans

Iaccarino G, Leosco D, Koch WJ. Eur Heart J 2005

Elevated myocardial and lymphocyte GRK2 expression and activity in human heart failure

>45% <45%0

10

20

30 *

Ejection Fraction(%)

Lym

phoc

yte

GR

K2

activ

ity(fm

olpi

/mg/

min

)

Lymphocytes Right Atriapt. 37 pt.53pt. 37 pt.53

0 20 40 60 800

10

20

30

40

y = 0.5741x - 0.4527R2= 0.5686, p<0.02

Lymphocytes GRK2(O.D.)

Rig

htA

ppen

dage

sG

RK

2(O

.D.)

GRK2

LymphocyteGRK2

Prior Post PriorPost PriorPost PriorPost

17.6 20.4 13.9 17.0

405060

p< 0.01

Priorβ-blocker

Postβ-blocker

0

3000

6000

9000

GR

K2

(D.U

.)

405060

p= NS

Priorβ-blocker

Postβ-blocker

0

10

20

30

VO2

max

(ml/K

g/m

in)

15.611.3

0.5

0.6

0.7

0.8

0.9

1.0

0 6 12 18 24 30 36Pr

opor

tion

aliv

e

Months

Responder

Non Responder

P< 0.01 (Log Rank Test)

Kaplan Meyer (adjusted)

Unpublished data

Different effects of β-blockers on lymphocyte GRK2levels in pts with heart failure

30

D.U

. (30

0 ug

IP)

GR

K2

Prior Post PriorPost PriorPost PriorPost

LymphocyteGRK2

17.6 20.4 13.9 17.0 12.6

405060

Priorβ-blocker

Postβ-blocker

0

3000

6000

9000

GR

K2

(D.U

.)

405060

p= NS

Priorβ-blocker

Postβ-blocker

0

10

20

30p= NS

The Role of β-adrenergic ReceptorDysregulation on Age-associated

Endothelial Dysfunction

Endothelium dependent β-AR vasorelaxation isimpaired in aged and hypertensive rats as well

Aging

Leosco D et al. Am J Physiol, 2003

-11 -10 -9 -8 -70.4

0.8

1.2

Isoproterenol Log M

Fold

of 1

0-6M

PE

*

-9 -8 -7 -6 -5 -40.0

0.4

0.8

1.2

Nitroprusside Log M

Fold

of 1

0-6M

PE

Endothelium independentvasorelaxation

β-AR vasorelaxation

youngold

old

young

Iaccarino G et al. Circulation, 2002

Hypertension

Isoproterenol Log M

Fold

of 1

0M

PE

β-AR vasorelaxation

Normotensive

Hypertensive

Nitroprusside Log M

Fold

of 1

0M

PE

Endothelium independentvasorelaxation

Young Old

Beta Binding

Young Old0

10

20

fmol

/mg

*

GRK2 Activity

Young Old 0

2500

5000

7500

10000

Arb

itrar

yU

nits *

OldGRK2

Arb

itrar

yU

nits

Young

Normoten

sive

Young

Hypert

ensive

Norm

oten

sive

Norm

oten

sive

Hype

rtens

iveHy

perte

nsive

GRK2

Leosco D et al. Am J Physiol 2003 Gros R et al. J Clin Invest 1997

Endothelial GRK2 activity is increased withaging and hypertension

Aging Hypertension

Mixed endothelial β3-adrenoceptor agonist and α1-adrenoceptor antagonist properties of nebivolol

Rozec B et al. British J Pharmacol 2006

SalbutamolNebivololNebivolol + Butoxamine

Nitric oxide production Cytosolic free Ca2+ concentrationBaseline Nebivolol

Baseline Nebivolol + Butoxamine

Nebivolol induces endothelial β2-adrenergic receptor–mediated nitric oxide production

Broeders MA et al. Circulation 2000

The Role of β-adrenergic ReceptorDysregulation on Age and Heart Failure-related

Impairment of Angiogenesis

Young Sedentary Young Exercised

Old Sedentary Old Exercised

Age-related impairment of angiogenesis in hindlimb ischemia

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

OldSED

Cap

illar

yto

fiber

OldEX

YoungSED

YoungEX

P< 0.01

P< 0.01

Leosco D et al. J Gerontol A Biol Sci, 2007

Iemitsu M et al. Am J Physiol 2006

Aging induces downregulation of VEGF angiogenicsignaling cascade in hearts

Angiogenesis impairment in the post-ischemic HF

Leosco D et al. Cardiovascular Research 2008

HF Sedentary HF Exercised Total capillary density(capillare/mm2)

Capillaries

Arteriolar Lenght density(mm/mm3)

Arterioles

HF untreated

HF β1-AR blockade

ShamOperated

HFUntreated

HFβ1AR block

*

Septum

Border area

Cap

illarie

s/m

µ2

0

0,2

0,4

0,6

0,8

1

1,2

β1-AR selective blockade improvesangiogenesis in the post-ischemic failing heart

Zincarelli C, Rengo G, and Leosco D et al. AHA Scientific Sessions 2008

SHAM HF untreated HF β1-AR blockade

0

10

20

30

40

50

60

70

2

LV FS%

*

SHAM HF β1-AR blockHF

ShamOperated

β-ARs density

HFUntreat

HFβ1-AR block

*

pmol

/mg

of m

embr

ane

prot

ein

01020304050

607080

c-AMP production

HFUntreat

ShamOperated

*

pmol

/mg/

min

0

10

20

30

40

50

60

70

HFβ1-AR block

Cardiac GRK2

00,20,40,60,81

1,21,41,6

*A.U

HFUntreat

HFβ1-AR block

ShamOperated

Zincarelli C, Rengo G, and Leosco D et al. AHA Scientific Sessions 2008

β1-AR selective blockade improves β1-AR pathwayand LV remodelling in the post-ischemic HF

tAkt

pAkt

HF Ctrl HF β1-AR block

eNOS

peNOS

GAPDH

VEGF

p-A

kt/t-

Akt

0

0,5

1

*

HF Ctrl HF β1-AR block

0

0,7

1,4

p-eN

OS/

eNO

S

*

HF Ctrl HF β1-AR block

VEG

F/G

APD

H

0

0,2

0,4

HF Ctrl HF β1-AR block

*

HF Ctrl HF β1-AR block HF Ctrl HF β1-AR block

Selective β1AR blockade activatesVEGF/Akt angiogenic pathway in HF

Zincarelli C, Rengo G, and Leosco D et al. AHA Scientific Sessions 2008

Shyngosine-1-Phosphate receptors (S1P1)

• Member of the superfamily of G-protein coupledreceptors (such as β-adrenergic receptors)

• Essential for vasculogenesis

• Highly expressed in the heart

• Regulated by GRK2

GRK2 overexpression induces β-AR internalizationand S1P1 receptors degradation in human

coronary arteries endothelial cells

β-AR

Basal Isoproterenol+

GRK2 overexpression

S1P1r

Basal Isoproterenol+

GRK2 overexpression

Data in progress

Heart Failure

CACA CACA

CACA

CACACACA

CACA CACACACA

CACACACACACA

CACA CACA

CACA

CACACACA

CACA CACACACA

CACACACA

CACA CACA

CACA

CACA

CACACACA CACA

CACACACA

CACA

CACACACA

P

GRK2

CACACACA

P

GRK2

β-AR S1P1

contractilityvasodilation

angiogenesis

Cardiomyocytes and Endothelial Cells

Extracellular

Cytosol

Conclusions

• Cardiac and vascular β-adrenergic receptordysfunction represent a common finding of aging

• These abnormalities resemble those observed in heart failure and hypertension

• β-AR blockade has been shown to reversereceptorial and post-receptorial abnormalities of β-ARsignaling

ß-adrenergic blockers Old and Novel Mechanisms of action

Sympathetic activationHRMyocardial ischemiaBlood pressureArrhythmiasOxidative stress, proliferation, Apoptotisβ-AR density and function (“The paradox”)Endothelium-mediated vasodilationAngiogenesis

Sympathetic activationHRMyocardial ischemiaBlood pressureArrhythmiasOxidative stress, proliferation, Apoptotis

Conclusions

• Cardiac and vascular β-adrenergic receptordysfunction represent a common finding of aging

• These abnormalities resemble those observed in heart failure and hypertension

• β-AR blockade has been shown to reversereceptorial and post-receptorial abnormalities of β-ARsignaling

• Other issues have to be considered for elderly ptswith cardiovascular diseases

New ConceptG Protein Coupled Receptor Dimerization

NEAII

ATP cAMPPIP2 IP3DAG

AngiotensinReceptor

β Adrenergic Receptor

Myocyte contractility

Gαq

AII

Barki-Harrington et al. Circulation 2003

β-blockers Inhibit Ang II Stimulated MyocyteContractility

Valsartan Uncouples Agonist-Stimulated βARs and Acts Like a β-Blocker In Vivo

Gαs

Val

Barki-Harrington et al. Circulation 2003

The LabFranco RengoPasquale AbeteGianluca TestaGiuseppe RengoCarmela ZincarelliDavid Della MorteLuca GolinoFrancesca FortunatoDaniela FemminellaElena Avallone

Cattedra di GeriatriaUniversità di Napoli “Federico II”

Fondazione S. MaugeriIstituto di Telese/Campoli IRCCS

Precious coworkers

Walter J Koch PhiladelphiaHoward Rockman DurhamGuido Iaccarino Napoli