title: Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex.
abstract: Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.
authors.affiliation: The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA.
authors.forename: William
authors.initials: W
authors.lastname: Schlecht
authors.affiliation: The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA.
authors.forename: King-Lun
authors.initials: KL
authors.lastname: Li
authors.affiliation: The Environmental and Molecular Science Laboratory, Pacific Northwest National Laboratory, 3335 Innovation Boulevard Richland, WA 99354, USA.
authors.forename: Dehong
authors.initials: D
authors.lastname: Hu
authors.affiliation: The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA.
authors.forename: Wenji
authors.initials: W
authors.lastname: Dong
mesh_terms: D000818:Animals; D015764:Bepridil / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002316:Cardiotonic Agents / Q000737:chemistry / Q000378:metabolism*; D006835:Hydrazones / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D011724:Pyridazines / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D000077464:Simendan; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002316:Cardiotonic Agents; D006835:Hydrazones; D011724:Pyridazines; D014336:Troponin; D000077464:Simendan; C041648:pimobendan; D015764:Bepridil; D002118:Calcium
keywords: cardiac tropnin; drug screening; fluorescence spectroscopy