Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca2+-sensitivity and suppress the modulation of Ca2+-sensitivity by troponin I phosphorylation

Abstract

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca2+-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca2+-sensitivity when compared with donor heart troponin and the Ca2+-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca2+-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca2+-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca2+-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca2+-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca2+-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies

“…Alamut is a licensed software package available from Interactive Biosoftware (). To determine the robustness of our in silico predictions, we compared the effect on the splicing process of 10 previously reported intron mutations verified by in vitro/in vivo assay [19,20,21,22,23,24] with the outcome of our Alamut analysis, and obtained similar results (Supplementary Table S1). The results obtained with this predictor software for the novel intron variants are summarized in Table 2.…”

Epigallocatechin-3-Gallate Accelerates Relaxation and Ca2+ Transient Decay and Desensitizes Myofilaments in Healthy and Mybpc3-Targeted Knock-in Cardiomyopathic Mice

“…EGCg has been reported to decrease myofilament Ca 2+ sensitivity in three HCM models expressing either a TNNT2 or TNNI3 mutation (Tadano et al, 2010; Warren et al, 2015; Messer et al, 2016). To assess whether the EGCg effects in intact cells resulted from a decrease in myofilament Ca 2+ sensitivity, we measured force-pCa relationships in skinned ventricular trabeculae from WT and KI mice.…”

“…Proposed mechanisms were anti-oxidative, anti-inflammatory, vasorelaxant, and positive inotropic effects (Chyu et al, 2004; Lorenz et al, 2004; Ludwig et al, 2004). Furthermore, it was shown that EGCg lowered myofilament Ca 2+ sensitivity in a transgenic HCM mouse model expressing a human cardiac troponin T ( TNNT2 , cTnT) mutant (Tadano et al, 2010) and in HCM-associated human cardiac troponin I ( TNNI3 , cTnI) and cTnT mutants in a reconstituted acto-myosin system (Warren et al, 2015; Messer et al, 2016). However, the effects of EGCg were not evaluated in other HCM models associated with mutations in the thick filament of the sarcomere.…”

Cardiomyopathies and Related Changes in Contractility of Human Heart Muscle

“…A case has been made that loss of this functional modulation of Ca 2+ -sensitivity through phosphorylation of TnI may play a pivotal role in HCM and DCM development and progression. The experiments using reconstituted thin filamnets in the in vitro motility assay provide a strong support that blunted response is one of the common abnormalities seen for both DCM [76,99,100] and HCM mutations [101,102]. It is interesting, that the effect was observed even if pathogenic mutations were not in thin filaments but in other sarcomeric proteins (myosin and MyBP-C) which were not present in the in vitro test system [76,101].…”