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.
Highlights
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy and is usually associated with mutationsAbbreviations: DCM, Dilated Cardiomyopathy; EGCG, Epigallocatechin-3Gallate; HCM, Hypertrophic Cardiomyopathy; in vitro motility assay (IVMA), In vitro Motility Assay; kACT, Rate of force development; kREL, Rate of fast relaxation phase; nH, Hill Coefficient; troponin C (TnC), Troponin C; troponin I (TnI), Troponin I; troponin T (TnT), Troponin T; WT, wild-type.in sarcomeric proteins
We initially studied a TnT mutation in a unique cardiac muscle sample from a patient with HCM due to a homozygous mutation in TnT, K280N
We previously found that this pattern of results was typical of muscle from the interventricular septum of hypertrophic obstructive cardiomyopathy (HOCM) patients that generally had an uncoupled relationship between Ca2þ-sensitivity and TnI phosphorylation accompanied by very low levels of TnI phosphorylation [23]
Summary
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy and is usually associated with mutations. A recent study has shown that 11% of the identified mutations are in the proteins of the thin filament; actin, tropomyosin, troponin I (TnI), troponin C (TnC) and troponin T (TnT) [1]. It is generally found that HCM-causing mutations result in a 2e3 fold higher myofilament Ca2þ-sensitivity compared to normal heart muscle and this has been proposed to be necessary to trigger the symptoms of HCM: a hyper-contractile phenotype, heart muscle hypertrophy, myocyte disarray and fibrosis [2], the clinical manifestation of HCM is very variable, probably due to background genetic and environmental factors. A.E. Messer et al / Archives of Biochemistry and Biophysics 601 (2016) 113e120.
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