modificazioni età-correlate del signaling β-adrenergico e
TRANSCRIPT
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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”
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CARDIOMYOCYTE
extracellular
intracellularACβ-AR
CACA CACA
CACA
CACACACA
CACA CACA
CACACACA
CACA
CA
ATP cAMP
Gsαβγ
PKA+
contractility
Physiologic conditions
CA
Gsα
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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
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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”
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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
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β-AR function changes with age of subject in myocytes from non failing human ventricle
Davies CH, Ferrara N, Harding SE. Cardiovasc Res, 1996
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β-Adrenergic Receptor desensitizationThe role of GRK2 (β-ARK1)
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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).
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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
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β-AR blockade reduces cardiac GRK2 (β-ARK1)levels in normal hearts
Iaccarino G, Lefkowitz RJ and Walter J. Koch. Circulation 1998
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Harding et al. PNAS 2001
Targeting GRK2 (β-ARK1) dramatically increasessurvival in animal models of heart failure
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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
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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
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Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007
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Inhibition of adrenal GRK2 activity leads to reduction of circulating cathecolamines
Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007
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Lymperopoulos A, Rengo G, Koch WJ. Nature Medicine 2007
Inhibition of adrenal GRK2 activity leads to improved function of the failing heart.
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Pathophysiologic role of GRK2 and the therapeuticpotential of its inhibition in heart failure.
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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
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GRK2 as a Novel Biomarkerof Heart Failure in Humans
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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
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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
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The Role of β-adrenergic ReceptorDysregulation on Age-associated
Endothelial Dysfunction
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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
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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
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Mixed endothelial β3-adrenoceptor agonist and α1-adrenoceptor antagonist properties of nebivolol
Rozec B et al. British J Pharmacol 2006
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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
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The Role of β-adrenergic ReceptorDysregulation on Age and Heart Failure-related
Impairment of Angiogenesis
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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
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Iemitsu M et al. Am J Physiol 2006
Aging induces downregulation of VEGF angiogenicsignaling cascade in hearts
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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New ConceptG Protein Coupled Receptor Dimerization
NEAII
ATP cAMPPIP2 IP3DAG
AngiotensinReceptor
β Adrenergic Receptor
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Myocyte contractility
Gαq
AII
Barki-Harrington et al. Circulation 2003
β-blockers Inhibit Ang II Stimulated MyocyteContractility
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Valsartan Uncouples Agonist-Stimulated βARs and Acts Like a β-Blocker In Vivo
Gαs
Val
Barki-Harrington et al. Circulation 2003
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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