A Functional Investigation of Mutations Associated with Hypertrophic and Dilated Cardiomyopathy
Author(s)
Dyer, Emma Catherine
Type
Thesis
Abstract
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).
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).
Date Issued
2008-01
Date Awarded
2008-06
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Marston, Steve
Sponsor
British Heart Foundation
Creator
Dyer, Emma Catherine
Publisher Department
Cardiac Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)