Download - Translational Studies in Heart Failure
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Translational Studies in HeartFailure
Wally Koch, PhD.W.W. Smith Professor of Medicine
C CENTERENTER F FOROR T TRANSLATIONALRANSLATIONAL M MEDICINEEDICINE
George Zallie and Family Laboratory for Cardiovascular Gene Therapy
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Effective Model of Translational Research
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Non-Effective Translational Research Model
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Heart Failure (HF) Statistics
•Epidemic Proportions>400,000 new cases per year in U.S. (5 million total)
•Death-rate still IncreasingCAD down 49% - CHF up 64% in last 20 years
•Morbidity and Costs also High#1 cause of all hospitalizations – >$300 Billion per year
•Therapies not IdealImprovements but no truly effective therapy
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Translational Research
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Translational Research
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The G Protein-Coupled ReceptorKinases (GRKs)
Serine/ Threonine Kinases 3 classes: GRK1 (Rhodopsin Kinase), GRK7
GRK2 (βARK1), GRK3 (βARK2) GRK4, GRK5, GRK6
Additional Interacting Partners:Tubulin, Actin, α-actinin
Adapted from Pao CS and Benovic JL, Science's STKE 8 October 2002, pp. pe42
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αα βγβγ
dissociationdissociation
ACAC
ATPATP
cAMPcAMP
activationactivation
AgonistAgonist
ββ-AR-AR
ββARKARK
bindingbinding &&activationactivation
βγβγ
DesensitizationDesensitization
AgonistAgonist
βARKct
ββARKARK
ββARKARK
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Translational Research
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Koch et al., Science, 1995.
αMHC βARK1 or βARKct coding regioncoding region intronintron -polyApolyA ++promoterpromoter
SV40
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ββARKctARKct rescues several different murine rescues several different murinemodels of HFmodels of HF
Murine model Result of βARKct cross Reference
MLP-/- Knockout Complete functional rescue with restored βAR responsiveness 1
Transgenic Cardiac Rescue of cardiac function with smallerCSQ Overexpression cardiac dimension and also improved survival 2
Transgenic Cardiac Rescue of function, prevention of hypertrophyExpression of a Mutant and dimensions and improved exerciseMyosin Heavy Chain (HCM) tolerance 3
Transgenic Cardiac Hypertrophy preventedOverexpression of MCP-1 4
Transgenic Cardiac Only βAR signaling improved withOverexpression of dominant- no functional or mortality rescueNegative mutant of CREB 5(CREBA133) 1. 1. Rockman et al. Rockman et al. 1998 PNAS 95:7000-7005.1998 PNAS 95:7000-7005.
2. 2. Harding et al. Harding et al. 2001 PNAS 98:5809-5814.2001 PNAS 98:5809-5814.3. 3. Freeman et al. Freeman et al. 2001 J 2001 J ClinClin Invest 107:967-974. Invest 107:967-974.4. 4. KhouriKhouri et al. et al. 2002 J 2002 J AmerAmer CollColl CardiolCardiol 39:I-164. 39:I-164.55. . Eckhart et al. 2002 J Mol Cell Eckhart et al. 2002 J Mol Cell CardiolCardiol 34:669-677 34:669-677..
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Rockman et al, PNAS 95:7000,1998.
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βARKct Rescue of Survival in A Transgenic Mouse Model of HF
Harding et al., PNAS, 2001.
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Translational ResearchModel
FROM MOUSE to MAN
• The βARKct transgene was cloned intoreplication-deficient adenoviral vectors
• Attempt intracoronary gene transfer tothe hearts of larger animal models
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Intracoronary Adenoviral-mediatedMyocardial Delivery
Sub-selective coronary artery catheterization
RCARCA LCxLCx
Shah et al. Circulation 101:408-414, 2000.Shah et al. Circulation 101:408-414, 2000.
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Shah et al. Shah et al. CirculationCirculation 2001;103:1311-1316 2001;103:1311-1316
Sub-selective catheter-mediated deliveryof βARKct: Chronic HF model
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Large Animal Gene Therapy
- Cardiopulmonary bypass with RA - Cardiopulmonary bypass with RA cannulationcannulation - - CardioplegicCardioplegic arrest (30 min) arrest (30 min) gene delivery gene delivery - Global transgene expression at 7 days - Global transgene expression at 7 days
AntLV
LatLV
PostLV
AntRV
PostRV Liver
AntSept
NLLiver (+)
36 kDA
PostSept
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Clinical trialsClinical trials
Large animalLarge animalmodelsmodels
Small animalSmall animalmodelsmodels
TransgenicsTransgenics
Ex vivoEx vivo testing testing
TherapeuticTherapeuticDesignDesign
ClinicalClinicalobservationobservation
Translational Research
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Final Proof of Concept for the βARKct
Will βARKct be beneficial in failing humanmyocytes ?
Heart arrested with 1L Heart arrested with 1L cardioplegiacardioplegia and explanted. and explanted.
Coronary artery (LAD or graft) Coronary artery (LAD or graft) cannulatedcannulated and and perfusedperfusedwith with collagenasecollagenase..
Myocytes incubated on Myocytes incubated on MatrigelMatrigel®®-coated plates.-coated plates.
Treated with Adenovirus and single cell contractionTreated with Adenovirus and single cell contractionmeasured as well as measured as well as ββARAR signaling. signaling.
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dL/dTdL/dT relaxation relaxation
FailingFailing(n=5 patients;(n=5 patients;10 cells/condition)10 cells/condition)
Failing+BARKctFailing+BARKct(n=5 patients;(n=5 patients;10 cells/condition)10 cells/condition)
dL/dTdL/dT contraction contraction
**
00
1010
2020
3030
4040
5050
6060
BasalBasal ISO ISO
p<0.05p<0.05
µµm
/sec
m/s
ec
001010202030304040505060607070
BasalBasal ISO ISO
**p<0.05p<0.05
µµm
/sec
m/s
ecRestoration of Contractility and Restoration of Contractility and ββAR Function AR Function
by by ββARKctARKct in Failing Human Myocytes in Failing Human Myocytes
Human myocytes infectedHuman myocytes infectedwith with Adeno-GFP/BARKctAdeno-GFP/BARKct
Williams et al, Circulation 2004.
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Conclusions
Molecular Normalization or “MolecularRemodeling” of the βAR System via GRK
Inhibition is Beneficial in Heart Failure and aNovel Therapeutic Strategy. Synergistic with
current HF therapy with βAR antagonists.
Inhibition of GRK2 (βARK1) represents apotential new drug class, targeting βAR andother GPCR systems from “the inside out”
Gene Therapy with βARKct will be first but also a definite need for small molecule.
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Cardiomyocyte membrane
Extracellular compartment
Cytoplasmic compartment
β2AR α AC
1 β agonist stimulation of βARs
βγ
βARK1
2 Dissociation of Gs protein
3 Activation of AC ↑cAMP
4 βARK1 inhibition
direct AC activationor overexpression
βAR overexpression
Translocation of βARK1 andphosphorylation of β1ARsand β2ARs
PKA
CaCa2+2+ cycling/induction of contraction cycling/induction of contraction SERCA2a
PLB inhibitionS100A1
β1AR
Potential Targets for Heart FailurePotential Targets for Heart FailureGene TherapyGene Therapy
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Hurdles to Human Application
• Target validation present for βARKct as well asother targets (S100A1, SERCA, Adenylyl Cyclase)
• Choice of vector– Advanced Adenovirus vs. AAV (or Lentivirus?)
• Route of vector administration– Invasive vs. non-invasive (CPB, coronary cath. or intra-
ventricular)• Choice of patient population
– End-stage, -/+ LVAD ? Or Class III/V, post CPBdysfunction
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GRK2 as a NovelBiomarker for Heart
Failure
Another Translational Approach – Clinical Research
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Can GRK2 be a Biomarker forHuman HF?
• A biomarker for heart failure is much needed• More therapeutic tools are needed for the treatment of
this condition• Evidence available in animals indicating this molecule as
a key player in experimental HF where its levels areregulated by the activation of the sympathetic nervoussystem
• To be exported in human settings we need confirmationthat in HHF1) GRK2 is pathophysiologically relevant2) is dysregulated during the disease3) is GRK2 important for prognosis in HHF4) its reduction can be beneficial
• To answer these and more questions we need a way tomonitor cardiac GRK2 repeatedly over the time
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0 20 40 60 80
0
10
20
30
40
y = 0.5741x - 0.4527
R2= 0.5686, p<0.02
Lymphocytes(O.D.)
Right Appendages
(O.D.)
Lymphocytes Right Atria
pt. 37 pt.53pt. 37 pt.53
80kD
A B
Cardiac GRK2 Tracks with Levels Foundin White Blood Cells
Iaccarino et al., Eur Heart J, 2005
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Lymphocyte GRK2 NegativelyAssociated with Cardiac Function
0 20 40 60 80
0
10
20
30
40
50
y =-0.21784x + 19.265
R2= 0.193, p<0.02
Ejection Fraction(%)
Lymphocytes Soluble
GRK Activity
(fmol pi/mg/min)
>45% <45%
0
10
20
30 *
Ejection Fraction(%)
Lymphocytes Soluble
GRK Activity
(fmol pi/mg/min)
1 2 3 4
0
10
20
30
40
NYHA Class
Lymphocytes Soluble
GRK Activity
(fmol pi/mg/min
F=4.272, p<0.02 ANOVA
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If High GRK2 is Associatedwith Worsened Function - is
GRK2 Lowered WithTreatment ?
Is GRK2 Involved in Reverse Remodeling Associated with LVAD Treatment ?
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• ~3000 cardiac transplants performed per year, LVADs arecommonly used as a “bridge to cardiac transplant”.
• LV unloading by LVAD support leads towardnormalization of myocardial structure and function(“reverse remodeling”) including restoration of βARresponsiveness.
• Long-term LVAD support leads to enhanced survival inpatients not eligible for transplant compared to optimizedmedical treatment (REMATCH Trial, Rose, et al, NEJM,2001) …however, 1-year mortality ~50%.
GRKs and LVAD Support in Human Heart FailureGRKs and LVAD Support in Human Heart Failure
• Will LVAD support in HF induce significant changes in myocardial GRK2 expression and GRK activity to
support improved βAR responsiveness as a positivecomponent to reverse remodeling ?
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Myocardial Myocardial ββARK1 proteinARK1 protein
80 80 kDakDa
CoreCore LVLV CoreCore LVLV (+)(+)CoreCore LVLV CoreCore LVLV
Den
sito
met
ricD
ensi
tom
etric
uni
ts/
uni
ts/ u
gug p
rote
in
pro
tein
00
0.20.2
0.40.4
0.60.6
0.80.8
11
1.21.2
1.41.4
**p<0.005p<0.005
GRK2 Levels After Mechanical Unloading in the GRK2 Levels After Mechanical Unloading in the Failing Human HeartFailing Human Heart
pico
gram
s R
NA
00
0.20.2
0.40.4
0.60.6
0.80.8
11
1.21.2
**p<0.05p<0.05
Pre-Pre-LVADLVADN=12N=12
Myocardial βARK1 mRNA
80 kDA
Z.C.Pre
Z.C.Post
I.B.Pre
I.B.Post
(+)
Post-Post-LVADLVADN=12N=12
Post-Post-LVADLVADN=12N=12
Pre-Pre-LVADLVADN=12N=12
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Cardiac (LV) GRK2 Tracks with LevelsFound in White Blood Cells
GRK2 LV vs lymphocyte (pre -LVAD)
30 40 50 60 70 80 900
1
2
3
Lymphocyte GRK2Densitometric units/300 ug protein (IP)
LV
GR
K2
De
ns
ito
me
tric
un
its
/ug
pro
tein
r2: 0.6665
p= 0.0251
GRK2 LV vs lymphocyte (post -LVAD)
20 25 30 35 40 45 50
0.0
0.2
0.4
0.6
0.8
1.0
Lymphocyte GRK2Densitometric units/300 ug protein (IP)
LV
GR
K2
De
ns
ito
me
tric
un
its
/ug
pro
tein
r2: 0.7503
p= 0.0117
Hata et al., JCF, 2006
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Summarizing GRK2 (βARK1) and GRK Activity asa Novel Biomarker in HF
• Alterations in GRK2 expression and GRKactivity seen in failing myocardium mirroredin lymphocytes and appears to beassociated with severity of disease anddecreased GRK2 associated with cardiacfunctional improvement.
• Potential for GRK2 levels and GRK activityin lymphocytes to be used as a biomarker inHF (surrogate marker for response totherapy currently being tested).
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Active Clinical Studies at JeffersonDoes Lymphocyte GRK2 Represent a Novel Biomarker for HF
Measurement of lymphocyte GRK2 and BNP innewly diagnosed HF patients. Temporal
assessment after β-blocker therapy.Correlation with treatment response.
Measurement of lymphocyte GRK2 in acutelydecompensated (hospitalized) HF patients anc
comparison to BNP for acute volumereduction.
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ACKNOWLEDGMENTS– Collaborators
Andrea Eckhart – JeffersonDavid Whellan _ JeffersonPaul Mather – JeffersonTerry Hyslop - Jefferson
Hugo Katus – HeidelbergJorg Heierhorst – Melbourne
Carmelo Milano – DukeHoward Rockman – DukeBob Lefkowitz - Duke
- SupportNIH/NHLBIGenzyme
– Koch LabKurt ChuprunSven PlegerPatrick MostJeff MartiniErhe GaoBrent DeGeorgeNatalie PatchMaggie ShapiroLiz MandelMatt KuhnWiebke PlegerTasos LymperopoulosMatt WilliamsJonathan HataAmit MittalNoah BloomgardenMatthieu Boucher