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Published on: February 13, 2012
Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex
William Schlecht1, King-Lun Li1, Dehong Hu2
1The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, PO Box 646515, Washington State University, Pullman, WA 99164-6515, USA.
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.
Insights
A new fluorescence assay reveals how calcium sensitizers work in heart failure. Different drugs like levosimendan and bepridil affect cardiac troponin C (cTnC) in unique ways, clarifying their mechanisms.
Area of Science:
- Cardiovascular Pharmacology
- Biochemistry
- Molecular Cardiology
Background:
- Calcium sensitizers are crucial for treating heart failure by improving cardiac contractility.
- The precise mechanisms of action for most calcium sensitizers remain poorly understood.
- Understanding these mechanisms is key to developing more effective heart failure therapies.
Purpose of the Study:
- To develop and validate an efficient fluorescence-based screening method to elucidate calcium sensitizer mechanisms.
- To investigate the effects of EMD 57033, levosimendan, bepridil, and pimobendan on cardiac troponin C (cTnC) function.
- To determine the context-dependent effects of these drugs on Ca(2+) sensitivity and relaxation kinetics.
Main Methods:
- Developed a novel fluorescence-based assay monitoring the hydrophobic cleft of cardiac troponin C (cTnC).
- Utilized Ca(2+) titration experiments to assess changes in cTnC Ca(2+) sensitivity and cooperativity.
- Employed stopped-flow experiments to analyze the impact of drugs on cTnC relaxation kinetics.
- Investigated drug effects in the presence of a cardiac troponin T (cTnT) phosphorylation mimic (T204E).
Main Results:
- Bepridil consistently increased cTnC Ca(2+) sensitivity across conditions.
- Levosimendan and pimobendan slowed cTnC closing, stabilizing its open conformation.
- Bepridil accelerated cTnC relaxation kinetics.
- All tested drugs (levosimendan, bepridil, pimobendan) increased Ca(2+) sensitivity in samples with the cTnT(T204E) mimic.
Conclusions:
- The developed fluorescence assay is effective for screening calcium sensitizers and elucidating their mechanisms.
- Calcium sensitizer effects on cTnC are drug-specific and can be context-dependent.
- These findings provide new insights into the molecular actions of key heart failure drugs, particularly in specific phosphorylation states.
