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Title: Structural and functional effects of troponin C cardiomyopathy mutations studied by polarized fluorescence
Author: Sevrieva, Ivanka Radkova
ISNI:       0000 0004 5368 1452
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2014
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Ca2+ binding to cardiac troponin C (cTnC) triggers a series of changes in thin filament structure that precede each heart contraction. Polarized fluorescence was employed to study the in situ structural and functional perturbations caused by two cardiomyopathy mutations in cTnC: L29Q associated with hypertrophic cardiomyopathy (HCM) and G159D associated with dilated cardiomyopathy (DCM). Native cTnC residues were replaced by a pair of cysteines either along the C- helix in the regulatory head domain of troponin or along the E-helix in the IT arm. The cysteines were cross-linked by a bifunctional rhodamine dye and the fluorescent proteins were passively exchanged in demembrenated rat ventricular trabeculae replacing ~80% of the endogenous cTnC. The orientation changes of the labelled helices were measured by polarized fluorescence simultaneously with generated tension as a function of [Ca2+]. L29Q and G159D did not affect maximum force at saturating [Ca2+]. Changes in C- and Ehelix orientation had the same steep Ca2+ dependence as active force production (Hill coefficient nH~4). Both missense mutations resulted in a very similar Ca2+ dependence of force when incorporated in E-helix labelled cTnCs and reduced Ca2+ sensitivity of force when incorporated in C-helix labelled cTnCs. This was most likely due to additive probe effects. L29Q has been linked to uncoupled Ca2+ sensitivity response to cTnI Ser22/Ser23 phosphorylation downstream of β-adrenergic signalling. Therefore, the functional effects of this mutation may be benign in the context of high Ser22/Ser23 phosphorylation background in our experimental system (~70% bisphosphorylated cTnI). Complete force inhibition by 25 μM blebbistatin decreased the Ca2+ sensitivity of C- and E-helix structural change. L29Q abolished this Ca2+ desensitizing effect of blebbistatin which suggests perturbed cross-bridge effects. There was an additional component of C-helix structural rearrangement at subthreshold [Ca2+] for both mutations, which lead to a different baseline for the Ca2+-dependent orientation change. Despite its location in the "structural" C-lobe of cTnC, G159D was able to induce orientation changes in the regulatory lobe and impact contractility. In the presence of G159D, E-helix orientation was offset over the entire range of [Ca2+], which might imply altered configuration of the IT arm.
Supervisor: Avkiran, Metin ; Sun, Yin-biao Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available