National Heart and Lung Institute, Imperial College London
Doi
Abstract
The contractility of human heart samples from patients diagnosed with hypertrophic
cardiomyopathy were studied using a quantitative in vitro motility assay. The aim of
this work was to investigate the molecular phenotype of thin filament proteins in the
HCM heart. Three biopsy samples with thin filament mutations were studied
alongside samples acquired from a subset of HCM patients classified with
hypertrophic obstructive cardiomyopathy.
The primary effect of thin filament mutations was investigated by reconstituting Factin
with ACTC E99K into thin filament with donor troponin. The E99K actin
filaments had a higher Ca2+-sensitivity then filaments composed of donor F-actin
(with no mutation) (EC50 E99K/donor 0.78±0.14, p=0.02). A similar higher Ca2+-
sensitivity was found when recombinant TnT K273N was incorporated into donor
troponin and compared to native donor troponin (EC50 K273N/donor 0.54±0.17,
p=0.006).
Troponin was also purified from HOCM heart samples. This troponin did not contain
a causative mutation but behaved abnormally in the response of thin filament Ca2+-
sensitivity to changes in TnI phosphorylation (EC50 PKA-HOCM/HOCM 1.08±0.25,
p=0.3) as mean TnI phosphorylation of PKA-HOCM was 1.56 molsPi/molsTnI and
HOCM was 0.29 molsPi/molsTnI. Thus, thin filament Ca2+-sensitivity was uncoupled
from TnI phosphorylation in thin filaments with HOCM troponin. When the native
TnT subunits were replaced with recombinant TnT this coupling was restored (EC50
HOCM rTnT/HOCM 0.63±0.26, p=0.03).
It would appear that the result of HCM-causing mutations are two-fold. The primary
effect of the HCM-causing mutations is to increase thin filament Ca2+-sensitivity.
However, the contraction machinery appears to be the target of secondary
modifications, that occur due to the pathology of the disease. Resulting in further
changes, such as changes in protein composition and post-translational modification.
One major consequence of these modifications may be to uncouple the relatively
labile regulation of thin filament Ca2+-sensitivity by TnI phosphorylation