The HCM-Associated Cardiac Troponin T Mutation K273N in a Human Heart Sample Studied by in Vitro Motility Assay

Abstract

We isolated troponin from left ventricular muscle of a patient with a homozygous K273N mutation in troponin T and compared it with troponin from donor heart muscle using the quantitative in vitro motility assay. Motility of thin filaments reconstituted with skeletal muscle actin, human tropomyosin and mutant native troponin was indistinguishable from thin filaments containing donor heart troponin (Sliding speed at 4µM Ca2+, K273N/donor = 0.98±0.09, n=9; Ca2+- sensitivity, EC50 fraction filaments motile K273N/donor = 0.94±0.28, n=9).This pattern of results was also observed with troponin from human HCM muscle caused by mutations in myosin and MyBP-C and in this case it was ascribed to a secondary modification of troponin not related to the mutation. To test for this possibility with our K273N mutant sample, we exchanged recombinant K273N TnT into troponin from a donor heart. In the non-HCM context the K273N mutation caused a substantial increase in Ca2+-sensitivity (EC50 fraction filaments motile K273N/donor = 0.54±0.17, n=5, p=0.004). We also exchanged recombinant K273N TnT into troponin from a patient with HCM due to a mutation in MyBP-C.We conclude that the K273N TnT mutation increases thin filament Ca2+-sensitivity, in common with other HCM-causing mutations, but in the heart muscle of patients with HCM this effect is largely masked by secondary changes in troponin function due to post-translational modifications. We are investigating whether troponin I phosphorylation is uncoupled from decreased Ca2+-sensitivity in this sample, since this dysfunction was observed in troponin from HCM patients with MYH7 and MYBPC3 mutations.Supported by Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577.