P118The HCM-associated cardiac Troponin T mutation K280N causes reduced responsiveness to protein kinase A

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Abstract

Background

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Mutations in cardiac Troponin T (cTnT) account for ∼15% of known HCM-causing mutations and are associated with a high incidence of sudden cardiac death at young age. In the present study we investigated myofilament properties in failing human myocardium with a homozygous cTnT charge mutation (K280N), obtained during transplant surgery. Troponin exchange experiments were performed in single cells to study the specific effect of the K280N mutation.

Methods

Force was measured in cardiomyocytes before and after exchange of endogenous mutant troponin with recombinant (healthy) human troponin at various calcium concentrations. To investigate myofilament responsiveness to β-adrenergic stimulation force measurements were repeated after incubation with protein kinase A (PKA).

Results

Maximal and passive force were significantly lower in K280N 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 K280N (pCa50 = 5.58 ± 0.02) compared to control (pCa50 = 5.52 ± 0.02). Myofilament response to PKA was smaller in K280N (ΔpCa50 = 0.03 ± 0.02) compared to non-failing (ΔpCa50 = 0.06 ± 0.01) cells. Partial exchange (∼70%) of endogenous K280N in HCM cells with healthy troponin did not correct the high myofilament Ca2+-sensitivity to control values, while myofilament responsiveness to PKA was significantly enhanced (ΔpCa50 = 0.17 ± 0.02). Protein analysis revealed lower cTnT phosphorylation in K280N 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 K280N in comparison with non-failing tissue. Phosphorylation of myosin light chain 2 was significantly higher in K280N compared to non-failing heart muscle.

Conclusions

Our data reveal reduced maximal force generating capacity and increased Ca2+- sensitivity of human myofilaments harbouring the HCM-associated cTnT mutation K280N. 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.

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