Familial Hypertrophic Cardiomyopathy (FHC)-Related Cardiac Troponin C (cTnC) L29Q Mutation Alters the Contractility and the Functional Effects of the Phosphomimetric cTnI

Abstract

The FHC-related cTnC mutation, L29Q, is located at the dysfunctional binding site I of cTnC and interacts with the N-terminus of cardiac troponin I (cTnI) during muscle contraction. Recent studies suggest that the L29Q cTnC mutation affects Ca2+ activation and the transduction of PKA-dependent phosphorylation (S23/24) cTnI effect on cTnC, but the results are contradictory. Therefore, to investigate whether the L29Q cTnC mutation abrogates the effects of PKA-dependent phosphorylation of cTnI of the Ca2+ sensitivity of cTnC, we examined the Ca2+ binding properties in multiple biochemical systems of increasing complexity. Mouse cTn was reconstituted with phosphomimetic cTnI (S23/24D), cTnT, and IAANS-labeled control TnC (T53C, C84S, C35S) ± the L29Q mutation. Steady-state Ca2+ binding and Ca2+ dissociation rates were measured in the isolated cTn as well as the reconstituted thin filaments. There was no significant difference between steady-state Ca binding for the control cTn (0.98±0.07μM) and L29Q cTn (0.99±0.08μM), nor a difference in the Ca2+ dissociation kinetics of reconstituted thin filaments, control (300±10/s) and L29Q (280±10/s). However, L29Q cTnC in skinned cardiac myocytes increased Ca sensitivity by 1.2 fold and reduced the decrease in Ca2+ sensitivity caused by phosphomimetic cTnI by ∼ 45%, as well as the enhanced length dependent Ca2+ sensitivity. These results suggest that the Ca2+ association rate, rather than the dissociation rate was affected by L29Q in the reconstituted thin filament. In conclusion, the cTnC L29Q mutation increased Ca2+ sensitivity of force generation and exacerbated the functional effects of TnI phosphorylation at S23/24 in skinned cardiac myocytes, but did not alter changes in the Ca2+ dissociation rate in the reconstituted thin filament caused by phosphomimetic cTnI.