Abstract 10889: Treating Heart Failure With Preserved Ejection Fraction Through Troponin I Phospho-mimicry

Abstract

Background —Heart failure with preserved ejection fraction (HF-pEF), characterized by resistance to ventricular filling from impaired myofilament relaxation (lusitropy), is the most common variant of heart failure. In HF-pEF, blunting of the lusitropic effect of troponin I (TnI) Ser-23/24 phosphorylation through the β-receptor-cAMP- protein kinase A (PKA) pathway contributes to impaired diastolic relaxation. Methods and Results —To identify small molecule compounds capable of generating the lusitropic effect of PKA-dependent TnI phosphorylation, we developed a 2D in vitro screen, where dye-labeled thin filaments are titrated with Ca 2+ and myosin motors. Thin filament activation is monitored by a FRET assay with FRET donor on the mobile element of TnI and the FRET acceptor on the C-lobe of troponin C (TnC). We observed that full activation of the thin filament required myosin binding. The Ca 2+ -sensitizer bepridil acted as myosin, enhancing thin filament affinity for Ca 2+ and stabilizing the association of the mobile element of TnI with TnC. In the presence of saturating bepridil, adding myosin had no effect on activation, suggesting that bepridil and myosin activate the thin filament through a common mechanism. PKA-treatment decreased the sensitivity of the thin filament to Ca 2+ and to myosin motors, and it reduced the FRET efficiency in the absence of Ca 2+ , suggesting that TnI phosphorylation causes a structural transition in the C-lobe of TnC. Epigallocatechin-3-gallate, the most prevalent polyphenol in green tea, acted as Ser23/24 phospho-TnI, reducing thin filament affinity for Ca 2+ and reducing affinity of myosin for the thin filament under both relaxing and Ca 2+ -activated conditions, suggesting that epigallocatechin-3-gallate and Ser23/24 phospho-TnI relax the myofilament through a common mechanism. Conclusions —Our results suggest that bepridil and myosin activate the cardiac thin filament through a common mechanism while epigallocatechin-3-gallate and Ser23/24 phospho-TnI deactivate the thin filament through a common mechanism. “Myofilament relaxers,” small molecule compounds that mimic the effects of Ser23/24 phospho-TnI may be an effective strategy in correcting diastolic dysfunction in HF-pEF.