Kinetic and Structural Characterization of Calcium Sensitizer Action on Thin Filament Function using FRET

Abstract

The use of novel calcium sensitizing agents in the treatment of heart disease offers therapeutic value for patients suffering from a particularly prevalent and recalcitrant condition, however the elusive mechanisms of action for these drugs prevent increased and improved utilization of such agents clinically. Ideally the calcium sensitizer impact on the thin filament could be monitored directly in a physiologically relevant and dynamic way while still capturing the molecular level mechanisms involved. With that aim a homo-FRET scheme was developed which monitors the N-domain opening of the calcium binding subunit of cardiac troponin (N-cTnC) by labeling with TAMRA at cTnC(cys-13) and cTnC(cys-51). Using this novel FRET design the calcium binding properties of reconstituted troponin and their deactivation kinetics were measured in the presence of calcium sensitizers Levosimendan, Bepridil, Pimobendan, and EMD-57033 at various levels of in vitro reconstitution. We hypothesized that depending on the mechanism for each calcium sensitizer the effects on cTnC calcium binding and deactivation kinetics would vary based on the level of reconstitution. We expect that new insight into which thin filament proteins are necessary for sensitizer action will yield a clearer picture of the molecular level mechanisms underlying cardiotonic action. Although the study is currently ongoing preliminary results show significant sensitization for all four drugs in reconstituted ternary troponin compared to control and that this effect is abrogated in samples containing only cTnC and cardiac troponin inhibitory subunit (cTnI). Deactivation kinetics show a decreased transition rate for Levosimendan, Bepridil and Pimobendan but not for EMD-57033 both at the ternary troponin level and at the cTnI-cTnC level. Results from measurements with reconstituted thin filament preparation will be also discussed.