P546Functional investigation of troponin with the homozygous HCM mutation, TNNT2 K280N, obtained from an explanted heart

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An explanted heart muscle sample from a patient previously diagnosed with hypertrophic cardiomyopathy was found to contain a homozygous TNNT2 K280N mutation.


We isolated troponin from this muscle sample; the presence of the K280N mutation and absence of wild-type sequence was established by mass spectroscopy. We compared its Ca2+-regulatory properties with donor heart troponin using the quantitative in vitro motility assay. Parallel investigations of contractility were made in skinned myocytes and in isolated myofibrils. Troponin phosphorylation was measured using phosphate affinity SDS-PAGE


Troponin I phosphorylation level in the K280N troponin was 1.43 ± 0.02 molsPi/mol compared with 1.57 ± 0.03 in donor heart. The maximum sliding speed of K280N- containing thin filaments and the Ca2+-sensitivity of the fraction of filaments motile were indistinguishable from donor heart troponin (Vmax K280N/Donor = 1.03 ± 0.11 ( n=8, p=0.41) , EC50 K280N/donor= 1.02 ± 0.19 (n=5, p=0.81)). When we exchanged K280N mutant recombinant troponin T into troponin from donor heart , thin filaments had a higher Ca2+-sensitivity than donor heart troponin (EC50, exchanged K280N/donor=0.54 ± 0.17, p=0.004). Exchange of wild-type troponin T into donor heart troponin had no effect on Ca2+-sensitivity.


We conclude that the K280N mutation in troponin T causes a higher Ca2+-sensitivity when studied in a non-failing heart context. This is typical of HCM-causing mutations and may be sufficient to trigger HCM. In the patient sample the Ca2+-sensitising effect of the K280N mutation was masked by secondary changes that could be related to the observed reduced sensitivity of the Ca2+-sensitivity to PKA-dependent phosphorylation.

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