Reduced Maximal Force and Increased Ca 2+-Sensitivity of Human Myofilaments Harbouring the HCM-Associated Cardiac Troponin T Mutation K273N

Abstract

Background Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. The main target of the thin filament is cardiac Troponin T (cTnT) accounting for ∼15% of the known HCM-causing mutations. Studies with recombinant proteins and transgenic animals showed that cTnT mutations increase Ca2+-sensitivity of the myofilaments. However, data in human myocardium are lacking.Methods We investigated sarcomeric function and protein phosphorylation in a HCM patient with a homozygous cTnT charge mutation (K273N). Myofilament force was measured at various calcium concentrations in demembranated single cardiomyocytes from the septum and left ventricular wall. To investigate myofilament responsiveness to β-adrenergic stimulation force measurements were repeated after incubation with exogenous protein kinase A (PKA). Protein composition was analysed by 1- and 2-dimensional gel electrophoresis.Results Maximal and passive force were significantly lower in K273N cells (21±2 and 1.9±0.1 kN/m2, respectively) compared to non-failing cardiomyocytes (33±4 and 2.9±0.4 kN/m2, respectively), while Ca2+-sensitivity was significantly higher in K273N (pCa50=5.58±0.02) compared to control (pCa50=5.52±0.02). K273N cardiomyocytes did not respond to PKA, while a significant reduction in Ca2+-sensititivity was observed in non-failing cells.Phosphorylation of cTnT was lower in K273N compared to non-failing myocardium and previously studied end-stage failing hearts. Phosphorylation of the PKA target proteins, cardiac myosin-binding protein-C was similar as found in non-failing myocardium, while troponin I phosphorylation was slightly lower in K273N.Conclusion Our data reveal reduced maximal force generating capacity and increased Ca2+-sensitivity of human myofilaments harbouring the HCM-associated cTnT mutation K273N. The lower force generating capacity and absence of myofilament Ca2+-desensitization upon PKA may impair cardiac function in human HCM with mutant cTnT at baseline and during exercise.Funding: Seventh Framework Program of the European Union “BIG-HEART,” grant agreement 241577.