1. The Myofilament Ca 2+ Sensitizer Levosimendan Preserves Systolic Function in Rats with Volume Overload Heart Failure Kristin Lewis, DVM Pathology Resident/Graduate Research Associate The Ohio State University, Columbus, OH The Research Institute, Nationwide Children’s Hospital, Columbus, OH

2. 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 Overload Pressure Overload

3. Volume Overload Progression of Volume Overload (VO) to Heart Failure Death Mitral regurgitation Systolic Dysfunction Diastolic Dysfunction HF LV RemodelingLV DysfunctionOvert HF Time (months to years)Time (months) ReversibleIrreversible Arterio-venous Fistulae

4. 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?

5. VO-induced HF with aortocaval fistula (ACF) in the rat Aorta 18g

6. Sham4 wk ACF ACF  progressive increase in LVEDd, LVEDs LVEDdLVEDs 15 wk ACF8 wk ACF Chest wall “Anterior” “Posterior” Time

7. VO is accompanied by functional deterioration % Fractional Shortening * * *= P < 0.05 vs. Sham LVEDdLVEDs

8. ACF  Altered Ca 2+ responsiveness and handling 8 wk ACF PLB pPLB SERCA2a ShamACF 8 wk ACF *** p<0.001 vs. Sham

9. Hypothesis Therapeutic strategies targeting myofilament Ca 2+ sensitivity will preserve/improve LV function in valvular heart disease

10. Myofilament Ca 2+ sensitizer: Levosimendan Hemodymanics Myocyte isolation Tissue 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.

11. Levo may attenuate the increase in LVEDD LVEDd LVEDs ShamACF-Veh ACF-Levo **** p<0.0001 vs Sham-Veh; ^ p<0.05, ^^ p<0.01 vs ACF-Veh

12. 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

13. Levo  ↑myofilament Ca 2+ sensitivity & ↑ maximal force without ↑ Ca 2+ transient * p<0.05, ** p<0.01 vs Sham-Veh ^ p<0.05, ^^^ p<0.001, ^^^^ p<0.0001 vs ACF-Veh

14. Levo does not result in vasodilation

15. Levo improved LV diastolic function **** p<0.0001 vs Sham-Veh ^ p<0.05, ^^^ p<0.001 vs ACF-Veh

16. cMyBP-C and cTnI Cardiac Myosin Binding Protein-C (cMyBP-C) – Thick filament associated protein – Phosphorylation  ↑ contraction and relaxation & ↓Ca 2+ sensitivity Cardiac Troponin I (cTnI) – Thin filament associated protein – Phosphorylation  ↓Ca 2+ sensitivity  earlier onset of relaxation Adapted from Landstrom AP, et al. Circulation. 2010 Dec 7;122(23):2441-9 Colson BA et al. J Mol Cell Cardiol. 2012 Nov; 53(5):609-16 Michalek AJ et al. Biophys J. 2013 Jan 22;104(2):442-52.

17. 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

18. Summary  Myofilament Ca 2+ sensitizer therapy  improved systolic and diastolic function  Improved systolic function is due to increased myofilament Ca 2+ sensitivity  Improved diastolic function may be due to cMyBP-C and/or cTnI phosphorylation  Myofilament Ca 2+ sensitizer therapy mildly attenuated increase in LVEDD  Therapeutic strategies targeting myofilament Ca 2+ sensitivity may improve function prior to load reduction surgery

19. 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