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Title: A functional investigation of mutations associated with hypertrophic and dilated cardiomyopathy
Author: Dyer, E. C.
ISNI:       0000 0000 7167 0985
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2008
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The mutations E361G and E99K in the cardiac actin gene cause dilated and hypertrophic cardiomyopathy respectively. Transgenic mouse models have been developed that over-express these mutations in their hearts. Mutant protein was expressed at 50% of to tal actin. Thin filaments were reconstituted with mouse actin, human cardiac tropomyosin and troponin, and studied by in vitro motility assay. Phosphorylated E361G and non-transgenic thin filaments were indistinguishable at all Ca2+-concentrations (EC50 NTg/E361G =1.07±0.11, p=0.59). E361G-actin reconstituted with dephosphorylated troponin had a lower Ca2+-sensitivity than non-transgenic (EC50 NTg/E361G = 0.47±0.09, p=0.01). When we compared E361G thin filaments containing phosphorylated and dephosphorylated troponin, the Ca2+-sensitivity was indistinguishable (EC50 E361G/E361G.dp = 1.03±0.08, p=0.74). This is in contrast to non-transgenic actin, where the EC50 of the phosphorylated thin filament was 3-times greater than dephosphorylated (p=0.003). The major functional change induced by the E361G mutation was therefore the abolition of the response to troponin phosphorylation. This would blunt the inotropic response in vivo and be the cause of the E361G DCM phenotype. The in vitro motility technique was extended to measure α-actinin binding affinity. The E361G mutant actin had a 5.3-fold reduced affinity for α-actinin, indicating a weaker attachment of filaments at the Z-line. Investigation of the DCM-causing mutation TNNC1 G159D in troponin isolated from human muscle showed a higher Ca2+ sensitivity with native phosphorylation levels (EC50 NF/G159D = 4.67±1.87) and upon dephosphorylation (EC50 NF/G159D = 1.77±0.29). G159D thin filaments also blunted the change in Ca2+-sensitivity when dephosphorylated (EC50 G159D/G159D.dp =1.24±0.17). The E99K mutation produced a strikingly different pattern of results to DCM mutations of an increased Ca2+-sensitivity and faster cross-bridge cycling rate, with phosphorylated (EC50 NTg/E99K = 2.54±0.64, p=0.07) and dephosphorylated troponin (EC50 NTg/E99K =1.2). The E99K mutation also 3 produced a reduced but not completely suppressed response to troponin phosphorylation (EC50 E99K/E99K.dp = 1.14±0.06, p=0.12).
Supervisor: Marston, Steve Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral