The Myofilament Ca2+ Sensitizer Levosimendan Preserves Systolic Function in

Rats with Volume Overload Heart Failure

Kristin Lewis, DVMPathology Resident/Graduate Research Associate

The Ohio State University, Columbus, OHThe Research Institute, Nationwide Children’s Hospital, Columbus, OH

2 types of hemodynamic overload HF

© Increased afterload© Concentric hypertrophy© Fibrosis© Examples:

• Hypertension• Aortic stenosis

© Increased preload© Eccentric hypertrophy© ECM degradation© Examples:

• Aortic/Mitral regurgitation • Myocardial infarct• Ventricular septal defect• Arterio-venous fistulae

Volume OverloadPressure Overload

VolumeOverload

Progression of Volume Overload (VO) to Heart Failure

Death

Mitral regurgitation

SystolicDysfunction

DiastolicDysfunction

HF

LV Remodeling LV Dysfunction Overt HF

Time (months to years) Time (months)

Reversible Irreversible

Arterio-venous Fistulae

MR treatment options

• Surgical repair/replacement– Optimal timing for patients with symptoms or decreased

function is defined– Optimal timing for asymptomatic patients is controversial

• Intervene early or “watch and wait”?

– Post-operative dysfunction

• Pharmacologic therapy– Can these agents delay surgery or improve function post-

operatively?– Optimal agents?

VO-induced HF with aortocaval fistula (ACF) in the rat

Aorta

18g

Sham 4 wk ACF

ACF progressive increase in LVEDd, LVEDs

LVEDd LVEDs

15 wk ACF8 wk ACF

Chest wall“Anterior”

“Posterior”

Time

VO is accompanied by functional deterioration

Sham ACF0

10

20

30

40

50

% Fractional Shortening

*

0 200 400 6000

50

100

150

Volume (l)

Pre

ssur

e (m

m H

g)

*

*= P < 0.05 vs. ShamLVEDd LVEDs % 𝐹𝑆=100 𝑥𝐿𝑉𝐸𝐷𝑑−𝐿𝑉𝐸𝐷𝑠

𝐿𝑉𝐸𝐷𝑑

-6.0 -5.5 -5.0 -4.5 -4.0

0

20

40

60

80

100

pCa

Fo

rce

(m

N/m

m2 )

Sham

ACF

*

ACF Altered Ca2+ responsiveness and handling

8 wk ACF

PLB

pPLB

SERCA2a

Sham ACF

8 wk ACF

*** p<0.001 vs. Sham

Arb

itra

ry U

nit

s

Serca 2a pPLB/PLB0

1

2

3 ShamACF

***

Hypothesis

Therapeutic strategies targeting myofilament Ca2+ sensitivity will preserve/improve LV

function in valvular heart disease

Myofilament Ca2+ sensitizer: Levosimendan

HemodymanicsMyocyte isolationTissue collection

(n=28)

(n=22)ACF

SHAM

ACF (n=23)

0 wk 8 wk

ECHO(q2w)

Levo, 1 mg/kg

Adapted from Papp Z, et al. Int J Cardiol. 2011 Jul 23.

Levo may attenuate the increase in LVEDD

LVEDd LVEDs

Sham ACF-Veh ACF-Levo

**** p<0.0001 vs Sham-Veh; ^ p<0.05, ^^ p<0.01 vs ACF-Veh

LVEDD

0 2 4 6 86

8

10

12

Study Week

(m

m) ****,̂ ^

********

****

********

****,^

Sham-VehACF-VehACF-Levo

Levo improved LV systolic function

* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 vs Sham-Veh^ p<0.05, ^^^^ p<0.0001 vs ACF-Veh

Fractional Shortening

0 2 4 6 825

30

35

40

45

50

Study week

%

**** ****

^̂ ^̂ ^̂ ^̂***

ACF-LevoACF-VehSham-Veh

Ees, Adj

mm

Hg

/uL

Sham-Veh ACF-Veh ACF-Levo0.0

0.2

0.4

0.6

0.8

1.0

***

*,̂

PRSW, Adj

mm

Hg

Sham-Veh ACF-Veh ACF-Levo0

50

100

150

*

^

Levo ↑myofilament Ca2+ sensitivity & ↑ maximal force without ↑ Ca2+ transient

-6.0 -5.5 -5.0 -4.5 -4.0

0

20

40

60

80

100

120

pCa

Fo

rce

(m

N/m

m2 )

ShamACF-Veh

ACF-Levo

**

^̂ ^

* p<0.05, ** p<0.01 vs Sham-Veh^ p<0.05, ^^^ p<0.001, ^^^^ p<0.0001 vs ACF-Veh

Peak[Ca2+]i

360

/380

Sham-Veh ACF-Veh ACF-Levo0

10

20

30*

^̂ ^̂

AS/PK

m*m

sec/

m

Sham-Veh ACF-Veh ACF-Levo0

20

40

60

*,̂

Peah h

t0

ACF-Veh

Sham-Veh

ACF-Levo

AreaC

Levo does not result in vasodilation

Mean arterial pressureP

ress

ure

(m

mH

g)

Sham-Veh ACF-Veh ACF-Levo0

50

100

150

Levo improved LV diastolic function

**** p<0.0001 vs Sham-Veh^ p<0.05, ^^^ p<0.001 vs ACF-Veh

Relaxation constantTau (W)

Sham-Veh ACF-Veh ACF-Levo0

5

10

15

mS

ec

^

dp/dtmin

Sham-Veh ACF-Veh ACF-Levo-10000

-8000

-6000

-4000

-2000

0

mm

Hg

/sec

****

^̂ ^

cMyBP-C and cTnI• Cardiac Myosin Binding

Protein-C (cMyBP-C)– Thick filament associated

protein– Phosphorylation ↑

contraction and relaxation & ↓Ca2+ sensitivity

• Cardiac Troponin I (cTnI)– Thin filament associated protein– Phosphorylation ↓Ca2+

sensitivity earlier onset of relaxation

Adapted from Landstrom AP, et al. Circulation. 2010 Dec 7;122(23):2441-9Colson BA et al. J Mol Cell Cardiol. 2012 Nov; 53(5):609-16Michalek AJ et al. Biophys J. 2013 Jan 22;104(2):442-52.

Phosphorylation at cMyBP-C Ser273, Ser302 and cTnI Ser23/24 may drive functional improvement

pSer273

Total cMyBP-C

Sham ACF ACF+L Sham ACF ACF+L

pSer302

Total cMyBP-C

Sham ACF ACF+L

pSer23/24

Total cTnI

cMyBP-C Ser273 Phosphoryation

Sham ACF-Veh ACF-Levo0.0

0.5

1.0

1.5

2.0

2.5

pS

er27

3/to

tal

*,̂ ^

cMyBP-C Ser302 Phosphorylation

Sham ACF-Veh ACF-Levo0

1

2

3

4

5

pS

er30

2/to

tal

*,̂

cTnI Ser23/24 Phosphorylation

Sham ACF-Veh ACF-Levo0.0

0.5

1.0

1.5

2.0

pT

nI/T

nI

*,̂ ^

Summary Myofilament Ca2+ sensitizer therapy improved systolic and

diastolic function

Improved systolic function is due to increased myofilament Ca2+ sensitivity

Improved diastolic function may be due to cMyBP-C and/or cTnI phosphorylation

Myofilament Ca2+ sensitizer therapy mildly attenuated increase in LVEDD

Therapeutic strategies targeting myofilament Ca2+ sensitivity may improve function prior to load reduction surgery

Acknowledgements

Nationwide Children’s Hospital• Lucchesi lab

– Pam Lucchesi– Aaron Trask– Aaron West– Jean Zhang– Anu Guggilam– Kirk Hutchinson– Mary Cismowski

• Vivarium– Natalie Snyder– Brenna Barbour– Erin Grove

The Ohio State University• Veterinary Biosciences

Funding Sources• ACVP/STP Coalition

Fellowship & Genentech• NIH R01-HL056046• Nationwide Children’s