Reversing HF: LVAD & clenbuterol LVAD with biological therapies for the reversal of HF. Myth or...

Reversing HF: LVAD & clenbuterol

LVAD with biological therapies for the reversal of HF.

Myth or reality?

Sir Magdi Yacoub, FRSImperial College London

Harefield Heart Science Centre

5th STEM CELL THERAPYMadrid 2008

Reversing HF: LVAD & clenbuterol• Chronology

– Beneficial and harmful effects of LVAD unloading

– ? optimal length of time for LVAD-induced functional recovery

– ? link between structural and functional reverse remodelling

– The role of clenbuterol

– Translational research (Harefield protocol)

– Clinical results

Reversing HF: LVAD & clenbuterol• Benefits of unloading

– Experimental (heterotopic Tx in murine models of HF)• Whole heart function• Cellular• Molecular• ECM

– Clinical• Bridge to Tx• Bridge to recovery


– Experimental (heterotopic Tx in murine models of HF)• Whole heart function

– in-vivo pressure-volume relationship of unloaded failing rabbit heart Tevaerai et al, JTCVS 2002

• Unloading improves contractile dysfunction

• restores cardiac responsiveness to mechanical and β-AR stimulation.



– in-vivo pressure-volume relationship of unloaded failing rabbit heart ± β2-AR overexpression Tevaerai et al, Circulation 2002

• Unloading improves contractile dysfunction

• β2-AR overexpression enhances functional improvement



– Ex-vivo pressure-volume relationship Soppa et al, Circulation 2006 (Abstract)

Maximum LV developed pressure

0 50 100 150 200 250 3000

50

100

150

Sham+Sal

HF+Sal

*

***

**

* *

** ***** ***

Preload (l)

Pre

ssu

re (

mm

Hg

)

0 50 100 150 200 250 3000

1000

2000

3000

4000

Sham+Sal

*** *

**

HF+Sal

**

*

*Maximum rate of LVpressure development

Preload (l)

Max

imu

m d

P/d

T(m

m H

g/s

)

Maximum LV developed pressure

0 50 100 150 200 250 3000

50

100

150

HF+Sal

HF+UN+Sal

Preload (l)

Pre

ssu

re (

mm

Hg

)

0 50 100 150 200 250 3000

1000

2000

3000

HF+Sal

HF+UN+Sal

Maximum rate of LVpressure development

Preload (l)

Max

imu

m d

P/d

T(m

m H

g/s

)

[A]

[B]

[C]

[D]


– Experimental (heterotopic Tx in murine models of HF)• Whole heart function• Cellular

– Cardiomyocyte size – Contractility– Ca2+ transient kinetics

• Molecular• ECM



– Cardiomyocyte size » 1 week - ↓21% in cell area Soppa et al, Circulation 2006 (Abstract)

» 2weeks – ↓23% in myocyte diameter Tsuneyoshi et al, ASAIO 2005

» 2 weeks – ↓27%, 4 weeks – ↓41%, 8 weeks – ↓56% in myocyte diameter Oriyanhan et al, JHLT 2007

– Contractility– Ca2+ transient kinetics




– Cardiomyocyte size – Contractility

» Time-dependent improvement followed by deterioration of papillary function and contractile reserve Oriyanhan et al, JHLT 2007

» Improved myocyte contractility after 2 weeks Takaseya et al, JACC 2004

» Unchanged myocyte contractility after 1 week Soppa et al, Circulation 2006 (Abstract)

– Ca2+ transient kinetics





» Normalization of deranged Ca2+ transient kinetics after 2 weeks unloading Takaseya et al, JACC 2004

» Unaffected deranged Ca2+ transient kinetics after 1 week unloading Soppa et al, Circulation 2006 (Abstract)





• Molecular– Gene and protein expression

» ↑SERCA2a Tsuneyoshi et al, ASAIO 2005; Takaseya et al, JACC 2004

» normalization of ↑BNP Tsuneyoshi et al, ASAIO 2005

» ↑β2-AR Tsuneyoshi et al, ASAIO 2005

• ECM


– Experimental (heterotopic Tx in murine models of HF)• Whole heart function• Cellular• Molecular• ECM

– ↑fibrosis Oriyanhan et al, JHLT 2007

Reversing HF: LVAD & clenbuterolHarmful effects of unloading

– Experimental (heterotopic Tx in murine models of HF)• Whole heart function – unchanged at 1 week

Soppa et al, Circulation 2006 (Abstract)

• Cellular– Contractility

» Unchanged at 1 week Soppa et al, Circulation 2006 (Abstract)

» Time dependent reduction in contractile function and contractile reserve Oriyanhan et al, JHLT 2007

– Ca2+ transient kinetics» Unchanged at 1 week Soppa et al, Circulation 2006 (Abstract)

• Molecular – not known• ECM

– ↑fibrosis Oriyanhan et al, JHLT 2007


– Clinical• Bridge to Tx

– Whole heart – improved ex-vivo LV function Klotz et al, Circulation 2005



– Whole heart» Improved LVEF, off-pump Frazier et al, ATS 1996; Muller et al, Circulation 1997

» Low incidence of myocardial functional recovery (4-24%) Frazier et al, ATS 1999; Mancini et al, Circulation 1998; Dandel et al, Circulation 2005

– Cellular» Reduction in cell size Zafeiridis et al, Circulation 1998; Bruckner et al, JHLT 2001

» Improved EC coupling


1. Harding et al., Circulation. 20012. Terracciano et al., Circulation. 2004.3. Chen et al., Circ Res. 20024. Marx et al., Cell 20005. Terracciano et al., Eur Heart J. 2003.6. Dipla et al., Circulation. 1998.7. Chaudhary et al., JACC. 2004.8. Frazier et al., ATS. 19969. Heerdt et al., Circulation. 2000

Improved EC coupling



– Whole heart– Cellular– Molecular

» Improved β-AR responsiveness and receptor density Dipla et al, Circulation 1998; Ogletree-Hughes et al, Circulation 2001

» ↑Ca2+ cycling gene & protein Heerdt et al, Circulation 2000

» Changes in cytokine milieu Torre-Amione et al, Circulation 1999

» Changes in gene transcription patterns Margulies et al, Circ Res 2005

» Improved mitochondrial function Heerdt et al, ATS 2002, Lee et al, JTCVS 1998

» Cytoskeletal protein changes Vatta et al, Lancet 2001 » Alterations in MAPK activity Flesch et al, Circulation 2001



– Whole heart– Cellular– Molecular– ECM - ? controversial

» ↓ collagen content Bruckner et al, JHLT 2001; Thompson et al, J Surg Res 2005; Akgul et al, JHLT 2004

» ↑collagen content Klotz et al, Circulation 2005; Li et al, Circulation 2001; Bruggink et al, JHLT 2006

» Alterations in MMPs & TIMPs Felkin et al, JHLT 2006; Klotz et al, Circulation 2005; Li et al,

Circulation 2001

Reversing HF: LVAD & clenbuterol• Chronology

– Beneficial and harmful effects of LVAD unloading

– ? optimal length of time for LVAD-induced functional recovery

– ? link between structural and functional reverse remodelling

– The role of clenbuterol

– Translational research (Harefield protocol)

– Clinical results

Reversing HF: LVAD & clenbuterol• ? optimal length of time for LVAD-induced

functional recovery

(Circulation. 2007;115:2497-2505)


SR Ca2+ content

Donor heart

Idiopathic dilated cardiomyopathy

Idiopathic dilated cardiomyopathy - post LVAD

• ? link between structural and functional reverse remodelling– specific changes in EC coupling, and not

regression of cellular hypertrophy Terracciano et al, Circulation 2004


Translational research (Harefield protocol)

“…. on its own, unloading appears to be inadequate. We therefore evolved a strategy of combination therapy, aiming at inducing maximal regression of pathological hypertrophy, followed by induction of physiological hypertrophy of both cardiac and skeletal muscle (using clenbuterol).”

Evolution of a concept

MH Yacoub



• Pharmacological therapy– Phase 1: intended to enhance reverse remodeling

• Digoxin

• ACE inhibition – SAVE, SOLVD, HOPE (Lisinopril)

• Angiotensin II inhibition - RESOLVD (Losartan)

• Aldosterone receptor blockade - RALES (Spironolactone)

• β-blockade (Carvedilol)



• Pharmacological therapy– Phase 2: intended to limit unloading-induced atrophy.

• β2 stimulation (Clenbuterol)


The role of clenbuterol

Reversing HF: LVAD & clenbuterolThe role of clenbuterol

• On skeletal muscle– ‘Physiological’ hypertrophy– Increased power Petrou et al, Circulation 1999; Maltin et al, Clin Sci (Lond) 1993

– Faster contraction & relaxation Petrou et al, Circulation 1999

– Induction of IGF1

• On cardiac muscle– ‘Physiological’ hypertrophy Wong et al, Card Res 1998; Petrou et al, Circulation 1995

– Enhancement of systolic & diastolic function in small & large animal models Wong et al, Card Res 1998; Hon et al, JTCVS 2001; Hon et al, Acta Physiol Scand 2001

– Prevents fibrosis following banding of the aorta & PA Hon et al, JTCVS 2001

– Gene expression (physiological hypertrophy) Wong et al, Card Res 1998; Petrou et al, Circulation

1995



• RV pressure-volume loops Hon et al, JTCVS 2001

Saline Clenbuterol



• Clenbuterol induces myofibrillogenesis

Clenbuterol 10µMUntreated


Clenbuterol induces an increase in the expression ofIGF-1 in cardiac myocytes in culture

Clen Iso PE Untreated0

1

2

3

4 ***

******

Mean + SEM: ***P<0.001 vs untreated cardiac myocytes(students t-test, unpaired)

Agonist (N=5)

Fo

ld in

cre

as

e in

IGF

-1 m

RN

Aa

bu

nd

an

ce

ov

er

co

ntr

ol (

Un

tre

ate

d=

1)


Reversing HF: LVAD & clenbuterol• Clenbuterol produces cardiac hypertrophy with altered EC coupling in

normal rat hearts Soppa et al, Am. J. Physiol, 2005

Cell surface area

Saline Clenbuterol3000

3500

4000

4500

5000p<0.05

mic

ron

2

saline clenbuterol

Clenbuterol prolongs APD

Am

plitu

de r

atio

(0.0

2 un

its)

Ca transient

Clenbuterol increases Ca

transient amplitude

100 ms

2000 ms

SR Ca content

Am

plitu

de r

atio

(0.0

2 un

its)

Clenbuterol increases

SR Ca content

Western blotting: clenbuterol

increases the expression of SERCA and

NCX


The role of clenbuterol•

Clenbuterol limits unloading-induced myocyte atrophy in normal and failing heart

Clen-Tx

Sal-Tx

Clen-Rec

Sal-Rec

50 µm50 m

Sham+Sal

HF+Sal

HF+Clen

HF+UN+Sal

HF+UN+Clen

Normal rat heart Soppa et al, Biophys J 2005 (Abstract)Failing rat heart Soppa et al, Circulation 2006 (Abstract)


Representative 2D and M-mode recordings showing development of LV dysfunction [A, B and C]. Graph [D] showing the effect of treatment with Sal or Clen on LVEF assessed by 2D short-axis views at the mid-papillary level.

0

10

20

30

40

50

60

70

80

90

***NS

NS

LV

EF

(%

)

HF+SalSham+Sal HF+Clen

[A] [B] [C]


• Clenbuterol improves LVEF of failing rat hearts Soppa et al, Circulation 2006 (Abstract)


0 50 100 150 200 250 3000

1000

2000

3000HF+Sal

HF+Clen

HF+UN+Sal

HF+UN+Clen*

* **

*§

§§

§

p<0.05; HF+UN+Clen vs HF+UN+Sal*§ p<0.05; HF+UN+Clen vs HF+Sal

Maximum rate of LVpressure development

Preload (l)

Max

imu

m d

P/d

T(m

m H

g/s

)


• Unloading+clenbuterol improves ex-vivo contractility of failing rat hearts Soppa et

al, Circulation 2006 (Abstract)


0.0 0.2 0.4 0.6 0.8 1.00.30

0.35

0.40

0.45

0.50

0.55

0.0 0.2 0.4 0.6 0.8 1.01.48

1.53

1.58

1.63

1.68

0.0

5 ra

tio u

nits

0.0

5 µ

m

200 ms

HF+Sal

HF+UN+Clen

0.0 0.2 0.4 0.6 0.8 1.00.30

0.35

0.40

0.45

0.50

0.55

0.0 0.2 0.4 0.6 0.8 1.01.48

1.53

1.58

1.63

1.68

Unloading+clenbuterol normalises EC coupling in normal & failing hearts

Unloading + Clen normalises delayed TTP shortening.

Sal Tx

Sal Rec

Clen Tx

Unloading + Clen normalises delayed sarcomere relaxation.

Unloading + Clen normalises delayed TTP

Normal rat heart Soppa et al, Biophys J 2005 (Abstract)

Unloading + Clen normalises increased Ca transient amplitude

Unloading + Clen normalises delayed decay Ca transient

Unloading + Clen normalises amplitude & speed of sarcomere shortening

Unloading + Clen normalises delayed Sarcomere relaxation

Failing rat heart Soppa et al, Circulation 2006 (Abstract)



• Clenbuterol normalises depressed myofilament sensitivity to Ca2+ of failing rat hearts, with and without unloading Soppa et al, Circulation 2006 (Abstract)

Myofilament sensitivity to Ca2+

0

1

2

3

* ****

Slo

pe (

m/r

ati

o u

nit

)



• Clenbuterol normalises unloading-induced reduction of Gαi protein expression Soppa et al, JMCC 2007 (Abstract)

Sham+Sal HF+Sal HF+Clen HF+UN+Sal HF+UN+Clen

45 kDa

Sham+S

al

HF+Sal

HF+Cle

n

HF+UN+Sal

HF+UN+Cle

n

0.0

0.1

0.2

0.3

0.4

0.5*

*

G

i (O

D n

orm

alis

ed t

o t

ota

l p

rote

in)



• β2-Gi sinalling has been shown to be cardioprotective Xiao et al, Trends Pharmacol Sci 2004

• Does clenbuterol activate the β2-Gi pathway?


Flu

o-4

flu

ore

sc

en

ce (

F/F

0)(r

.u.

0.2

) 500 ms

control 3x10-5 M 10-4 M wash out

clenbuterol

Sar

co

me

re l

eng

th (m

m)

wash out3 x10-5 M 10-4 M

1.9

1.8

##

#


• Acute clenbuterol application is –vely inotropic Siedlecka et al, AHA 2007 (Abstract No. 2492 )


-8 -7 -6 -5 -4control0

50

100

150

200

250 clenbuterol (n=6)

salbutamol (n=9)

fenoterol (n=9)

washout

**

***

##

## ###

###

###***

***

***

log [ 2-agonist] [M]

Co

ntr

acti

on

Am

pli

tud

e[%

of

con

tro

l]

-6 -5 -4control0

50

100

CLEN + CGP 20712A 300 nM (n=10)

CLEN + CGP 20712A 300 nM + ICI 118551 50 nM (n=10)

washout

# ##

###

###

###

CLEN (n=13)

**

#

######

######

###

log [clenbuterol] [M]

Co

ntr

acti

on

Am

pli

tud

e[%

of

con

tro

l]

-4.5 -4.0control0

50

100

150

CLEN + CGP 20712 nM (n=7)

washout

* ***

CLEN + CGP 20712 nM + PTX 2 g/ml (n=6)

###

###

log [clenbuterol] [M]

Co

ntr

acti

on

Am

pli

tud

e[%

of

con

tro

l]

The role of clenbuterol• Acute clenbuterol application is –vely inotropic and reduces contractility of

ventricular myocytes via a Gi-mediated pathway Siedlecka et al, AHA 2007 (Abstract No. 2492 )



• Explantation criteria

– LV dimensions with device off EDD <60mm, ESD <50 mm,

– EF >45%

– LVEDP <12 mmHg

– Resting CI >2.8 L/min/m2

– mVO2 >20 ml/kg/min & VE/VCO2 slope <34


Drug Therapy For Myocardial Recovery


Imperial CollegeOF SCIENCE TECHNOLOGY AND MEDICINE

Harefield HospitalRoyal Brompton & Harefield NHS Trust

20 Patients

5 Deaths (25%) 15 Survivors (75% comb ther)(4 Periop, 1 Late Infection)

4 Transplanted 11 Explanted (73%)(1 Perioperative Death)

1 Periop Death (7%)

1 Late death – lung Ca

Harefield Recovery StudyOutcome

1 Transplanted(after 2.5 years)

Reversing HF: LVAD & clenbuterolEjection Fraction Post Explantation

0

10

20

30

40

50

60

70

80

90

Months after Explantation

EF

(%)

Tx (n = 1)ExplantationBiV (n = 1)

Birks et al. N Engl J Med 2006;355:1873-84

Reversing HF: LVAD & clenbuterolmVO2 Post Explantation

05

1015202530354045

Months after Explantation

mV

O2

Birks et al. N Engl J Med 2006;355:1873-84

Reversing HF: LVAD & clenbuterol• Benefits of unloading+clenbuterol

– Clinical• Bridge to Recovery

– Whole heart» ↑LVEF and CI Birks et al NEJM 2006; Dandel et al, Circulation 2005; Frazier et al, ATS 1999

– Cellular» ↓size & normalised EC coupling Terracciano et al, Circulation 2004; Terracciano et al, EHJ 2003

– Molecular» Specific changes in cytoskeletal genes and proteins Latif et al, JHLT

2007; Birks et al, Circulation 2005 » Specific changes EPAC2, Integrin signalling pathway Hall et al, EHJ

2007

» AGAT – creatine synthesis Cullen et al, Circulation 2006

» ↑IGF-1 expression Barton et al, Circulation 2005

» ↑Apelin receptor expression Farkasfalvi et al, Circulation 2006 (Abstract)

– ECM – not known

Reversing HF: LVAD & clenbuterolConclusions

• Reversal of HF using LVAD + Clen in non-ischemic DCM has given encouraging results (single centre)

• The molecular & cellular mechanisms responsible have been partially studied experimentally & clinically

• A multicentre study of the ‘Harefield protocol’ (HARPS) is on the way

• Need to evaluate the combination therapy in – Ischemic DCM

– With continuous flow pumps


HARPS Investigator’s Meeting

Boston, MA April 10, 2008

Reversing HF: LVAD & clenbuterolStudy Site Status

INDIRB

Approval Contracts SIV#

Enrolled

Cleveland Clinic Not submitted Working on it

Michigan 2

Montefiore 2 (3)

Northwestern 2

Ohio State 2

Pennsylvania Conditional Submitted

Texas Heart 2

UAB ? Working on it Working on it

Washington 1

Reversing HF: LVAD & clenbuterolEnrollment Summary

0

5

10

15

20

25

30

35

40

45

Jun-

07

Jul-0

7

Aug

-07

Sep

-07

Oct

-07

Nov

-07

Dec

-07

Jan-

08

Feb

-08

Mar

-08

Apr

-08

May

-08

Jun-

08

Jul-0

8

Aug

-08

Sep

-08

Oct

-08

Nov

-08

Dec

-08

Jan-

09

Feb

-09

Mar

-09

Apr

-09

May

-09

Jun-

09

En

rollm

en

t

Actual

30 pts projected

40 pts projected

N=12 (includes pending MO & TX patients)

2 withdrawn

Reversing HF: LVAD & clenbuterol LVAD with biological therapies for the reversal of HF. Myth or...

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Transcript of Reversing HF: LVAD & clenbuterol LVAD with biological therapies for the reversal of HF. Myth or...