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Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-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 Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (?14, ?28 + 7, ?E160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-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 Ca(2+)-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.

SUBMITTER: Messer AE

PROVIDER: S-EPMC4909753 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

Messer Andrew E AE Bayliss Christopher R CR El-Mezgueldi Mohammed M Redwood Charles S CS Ward Douglas G DG Leung Man-Ching MC Papadaki Maria M Dos Remedios Cristobal C Marston Steven B SB

Archives of biochemistry and biophysics 20160329

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 Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sens ...[more]

PMID: 27036851

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Project description:Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

Project description:Recently four new hypertrophic cardiomyopathy mutations in cardiac troponin C (cTnC) (A8V, C84Y, E134D, and D145E) were reported, and their effects on the Ca(2+) sensitivity of force development were evaluated (Landstrom, A. P., Parvatiyar, M. S., Pinto, J. R., Marquardt, M. L., Bos, J. M., Tester, D. J., Ommen, S. R., Potter, J. D., and Ackerman, M. J. (2008) J. Mol. Cell. Cardiol. 45, 281-288). We performed actomyosin ATPase and spectroscopic solution studies to investigate the molecular properties of these mutations. Actomyosin ATPase activity was measured as a function of [Ca(2+)] utilizing reconstituted thin filaments (TFs) with 50% mutant and 50% wild type (WT) and 100% mutant cardiac troponin (cTn) complexes: A8V, C84Y, and D145E increased the Ca(2+) sensitivity with only A8V demonstrating lowered Ca(2+) sensitization at the 50% ratio when compared with 100%; E134D was the same as WT at both ratios. Of these four mutants, only D145E showed increased ATPase activation in the presence of Ca(2+). None of the mutants affected ATPase inhibition or the binding of cTn to the TF measured by co-sedimentation. Only D145E increased the Ca(2+) affinity of site II measured by 2-(4'-(2''-iodoacetamido)phenyl)aminonaphthalene-6-sulfonic acid fluorescence in isolated cTnC or the cTn complex. In the presence of the TF, only A8V was further sensitized to Ca(2+). Circular dichroism measurements in different metal-bound states of the isolated cTnCs showed changes in the secondary structure of A8V, C84Y, and D145E, whereas E134D was the same as WT. PyMol modeling of each cTnC mutant within the cTn complex revealed potential for local changes in the tertiary structure of A8V, C84Y, and D145E. Our results indicate that 1) three of the hypertrophic cardiomyopathy cTnC mutants increased the Ca(2+) sensitivity of the myofilament; 2) the effects of the mutations on the Ca(2+) affinity of isolated cTnC, cTn, and TF are not sufficient to explain the large Ca(2+) sensitivity changes seen in reconstituted and fiber assays; and 3) changes in the secondary structure of the cTnC mutants may contribute to modified protein-protein interactions along the sarcomere lattice disrupting the coupling between the cross-bridge and Ca(2+) binding to cTnC.

Dataset Information

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

Publications

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

Similar Datasets

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