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 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

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

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

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

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

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

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

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 Jul 23.

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

Levo improved LV systolic function * p<0.05, ** p<0.01, *** p<0.001, **** p< vs Sham-Veh ^ p<0.05, ^^^^ p< vs ACF-Veh

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< vs ACF-Veh

Levo does not result in vasodilation

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

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 Dec 7;122(23): Colson BA et al. J Mol Cell Cardiol Nov; 53(5): Michalek AJ et al. Biophys J Jan 22;104(2):

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

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

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-HL Nationwide Children’s