Translational Studies in Heart Failure

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

Clinical trialsClinical trials

Large animalLarge animalmodelsmodels

Small animalSmall animalmodelsmodels

TransgenicsTransgenics

Ex vivoEx vivo testing testing

TherapeuticTherapeuticDesignDesign

ClinicalClinicalobservationobservation

Effective Model of Translational Research

Non-Effective Translational Research Model

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








Translational Research

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

αα βγβγ

dissociationdissociation

ACAC

ATPATP

cAMPcAMP

activationactivation

AgonistAgonist

ββ-AR-AR

ββARKARK

bindingbinding &&activationactivation

βγβγ

DesensitizationDesensitization

AgonistAgonist

βARKct

ββARKARK

ββARKARK

Koch et al., Science, 1995.

αMHC βARK1 or βARKct coding regioncoding region intronintron -polyApolyA ++promoterpromoter

SV40

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..

Rockman et al, PNAS 95:7000,1998.

βARKct Rescue of Survival in A Transgenic Mouse Model of HF

Harding et al., PNAS, 2001.








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

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.

Shah et al. Shah et al. CirculationCirculation 2001;103:1311-1316 2001;103:1311-1316

Sub-selective catheter-mediated deliveryof βARKct: Chronic HF model








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

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.

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.

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.

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

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

GRK2 as a NovelBiomarker for Heart

Failure

Another Translational Approach – Clinical Research

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

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

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

If High GRK2 is Associatedwith Worsened Function - is

GRK2 Lowered WithTreatment ?

Is GRK2 Involved in Reverse Remodeling Associated with LVAD Treatment ?

• ~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 ?

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

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

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).

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.

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

Translational Studies in Heart Failure

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