Two mutations in troponin I that cause hypertrophic cardiomyopathy have contrasting effects on cardiac muscle contractility

2002 ◽  
Vol 362 (2) ◽  
pp. 443-451 ◽  
Author(s):  
David BURTON ◽  
Hassan ABDULRAZZAK ◽  
Adam KNOTT ◽  
Kathryn ELLIOTT ◽  
Charles REDWOOD ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Wild Type ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Inhibitory Function ◽  
Type Protein ◽  
Wild Type Protein

We investigated the effects of two mutations in human cardiac troponin I, Arg145 → Gly and Gly203 → Ser, that are reported to cause familial hypertrophic cardiomyopathy. Mutant and wild-type troponin I, overexpressed in Escherichia coli, were used to reconstitute troponin complexes in vanadate-treated guinea pig cardiac trabeculae skinned fibres, and thin filaments were reconstituted with human cardiac troponin and tropomyosin along with rabbit skeletal muscle actin for in vitro motility and actomyosin ATPase assays. Troponin containing the Arg145 → Gly mutation inhibited force in skinned trabeculae less than did the wild-type, and had almost no inhibitory function in the in vitro motility assay. There was an enhanced inhibitory function with mixtures of 10–30% [Gly145]troponin I with the wild-type protein. Skinned trabeculae reconstituted with troponin I containing the Gly203 → Ser mutation and troponin C produced less Ca2+-activated force (64±8% of wild-type) and demonstrated lower Ca2+ sensitivity [ΔpCa50 (log of the Ca2+ concentration that gave 50% of maximal activation) 0.25 unit (P < 0.05)] compared with wild-type troponin I, but thin filaments containing [Ser203]-troponin I were indistinguishable from those containing the wild-type protein in in vitro motility and ATPase assays. Thus these two mutations each result in hypertrophic cardiomyopathy, but have opposite effects on the overall contractility of the muscle in the systems we investigated, indicating either that we have not yet identified the relevant alteration in contractility for the Gly203 → Ser mutation, or that the disease does not result directly from any particular alteration in contractility.

scholarly journals Facilitated Cross-Bridge Interactions with Thin Filaments by Familial Hypertrophic Cardiomyopathy Mutations inα-Tropomyosin

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Fang Wang ◽  
Nicolas M. Brunet ◽  
Justin R. Grubich ◽  
Ewa A. Bienkiewicz ◽  
Thomas M. Asbury ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Molecular Mechanisms ◽  
Helical Structure ◽  
Familial Hypertrophic Cardiomyopathy ◽  
Maximum Speed ◽  
Sliding Speed ◽  
Thin Filaments ◽  
In Vitro Motility ◽  
Cardiac Sarcomeres

Familial hypertrophic cardiomyopathy (FHC) is a disease of cardiac sarcomeres. To identify molecular mechanisms underlying FHC pathology, functional and structural differences in three FHC-related mutations in recombinantα-Tm (V95A, D175N, and E180G) were characterized using both conventional and modified in vitro motility assays and circular dichroism spectroscopy. Mutant Tm's exhibited reducedα-helical structure and increased unordered structure. When thin filaments were fully occupied by regulatory proteins, little or no motion was detected at pCa 9, and maximum speed (pCa 5) was similar for all tropomyosins. Ca2+-responsiveness of filament sliding speed was increased either by increasedpCa50(V95A), reduced cooperativityn(D175N), or both (E180G). When temperature was increased, thin filaments with E180G exhibited dysregulation at temperatures ~10°C lower, and much closer to body temperature, than WT. When HMM density was reduced, thin filaments with D175N required fewer motors to initiate sliding or achieve maximum sliding speed.

Phosphorylation of threonine 276 in Smad4 is involved in transforming growth factor-β-induced nuclear accumulation

2003 ◽  
Vol 285 (4) ◽  
pp. C823-C830 ◽  
Author(s):  
Bernard A. J. Roelen ◽  
Ori S. Cohen ◽  
Malay K. Raychowdhury ◽  
Deborah N. Chadee ◽  
Ying Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Phosphorylation Site ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Mutant Protein ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Smad4, the common Smad, is central for transforming growth factor (TGF)-β superfamily ligand signaling. Smad4 has been shown to be constitutively phosphorylated (Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin C-H, Miyazono K, and ten Dijke P. EMBO J 16: 5353-5362, 1997), but the site(s) of phosphorylation, the kinase(s) that performs this phosphorylation, and the significance of the phosphorylation of Smad4 are currently unknown. This report describes the identification of a consensus ERK phosphorylation site in the linker region of Smad4 at Thr276. Our data show that ERK can phosphorylate Smad4 in vitro but not Smad4 with mutated Thr276. Flag-tagged Smad4-T276A mutant protein accumulates less efficiently in the nucleus after stimulation by TGF-β and is less efficient in generating a transcriptional response than Smad4 wild-type protein. Tryptic phosphopeptide mapping identified a phosphopeptide in Smad4 wild-type protein that was absent in phosphorylated Smad4-T276A mutant protein. Our results suggest that MAP kinase can phosphorylate Thr276 of Smad4 and that phosphorylation can lead to enhanced TGF-β-induced nuclear accumulation and, as a consequence, enhanced transcriptional activity of Smad4.

scholarly journals Mavacamten rescues increased myofilament calcium sensitivity and dysregulation of Ca2+ flux caused by thin filament hypertrophic cardiomyopathy mutations

2020 ◽  
Vol 318 (3) ◽  
pp. H715-H722 ◽  
Author(s):  
Alexander J. Sparrow ◽  
Hugh Watkins ◽  
Matthew J. Daniels ◽  
Charles Redwood ◽  
Paul Robinson
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Cardiac Troponin ◽  
Thin Filament ◽  
Troponin I ◽  
Troponin T ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Calcium Handling ◽  
Cellular Models

Thin filament hypertrophic cardiomyopathy (HCM) mutations increase myofilament Ca2+ sensitivity and alter Ca2+ handling and buffering. The myosin inhibitor mavacamten reverses the increased contractility caused by HCM thick filament mutations, and we here test its effect on HCM thin filament mutations where the mode of action is not known. Mavacamten (250 nM) partially reversed the increased Ca2+ sensitivity caused by HCM mutations Cardiac troponin T (cTnT) R92Q, and cardiac troponin I (cTnI) R145G in in vitro ATPase assays. The effect of mavacamten was also analyzed in cardiomyocyte models of cTnT R92Q and cTnI R145G containing cytoplasmic and myofilament specific Ca2+ sensors. While mavacamten rescued the hypercontracted basal sarcomere length, the reduced fractional shortening did not improve with mavacamten. Both mutations caused an increase in peak systolic Ca2+ detected at the myofilament, and this was completely rescued by 250 nM mavacamten. Systolic Ca2+ detected by the cytoplasmic sensor was also reduced by mavacamten treatment, although only R145G increased cytoplasmic Ca2+. There was also a reversal of Ca2+ decay time prolongation caused by both mutations at the myofilament but not in the cytoplasm. We thus show that mavacamten reverses some of the Ca2+-sensitive molecular and cellular changes caused by the HCM mutations, particularly altered Ca2+ flux at the myofilament. The reduction of peak systolic Ca2+ as a consequence of mavacamten treatment represents a novel mechanism by which the compound is able to reduce contractility, working synergistically with its direct effect on the myosin motor. NEW & NOTEWORTHY Mavacamten, a myosin inhibitor, is currently in phase-3 clinical trials as a pharmacotherapy for hypertrophic cardiomyopathy (HCM). Its efficacy in HCM caused by mutations in thin filament proteins is not known. We show in reductionist and cellular models that mavacamten can rescue the effects of thin filament mutations on calcium sensitivity and calcium handling although it only partially rescues the contractile cellular phenotype and, in some cases, exacerbates the effect of the mutation.

scholarly journals Heparin Induces Harmless Fibril Formation in Amyloidogenic W7FW14F Apomyoglobin and Amyloid Aggregation in Wild-Type Protein In Vitro

PLoS ONE ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. e22076 ◽  
Author(s):  
Silvia Vilasi ◽  
Rosalba Sarcina ◽  
Rosa Maritato ◽  
Antonella De Simone ◽  
Gaetano Irace ◽  
...  
Keyword(s):  
Fibril Formation ◽  
Amyloid Aggregation ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

Familial hypertrophic cardiomyopathy mutations in troponin I (K183Δ, G203S, K206Q) enhance filament sliding

2003 ◽  
Vol 14 (2) ◽  
pp. 117-128 ◽  
Author(s):  
Jan Köhler ◽  
Ying Chen ◽  
Bernhard Brenner ◽  
Albert M. Gordon ◽  
Theresia Kraft ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Troponin I ◽  
Isometric Force ◽  
Familial Hypertrophic Cardiomyopathy ◽  
In Vitro Assays ◽  
Maximum Speed ◽  
Exchange Method ◽  
In Vitro Motility ◽  
Maximum Isometric Force

A major cause of familial hypertrophic cardiomyopathy (FHC) is dominant mutations in cardiac sarcomeric genes. Linkage studies identified FHC-related mutations in the COOH terminus of cardiac troponin I (cTnI), a region with unknown function in Ca2+ regulation of the heart. Using in vitro assays with recombinant rat troponin subunits, we tested the hypothesis that mutations K183Δ, G203S, and K206Q in cTnI affect Ca2+ regulation. All three mutants enhanced Ca2+ sensitivity and maximum speed ( smax) of filament sliding of in vitro motility assays. Enhanced smax (pCa 5) was observed with rabbit skeletal and rat cardiac (α-MHC or β-MHC) heavy meromyosin (HMM). We developed a passive exchange method for replacing endogenous cTn in permeabilized rat cardiac trabeculae. Ca2+ sensitivity and maximum isometric force did not differ between preparations exchanged with cTn(cTnI,K206Q) or wild-type cTn. In both trabeculae and motility assays, there was no loss of inhibition at pCa 9. These results are consistent with COOH terminus of TnI modulating actomyosin kinetics during unloaded sliding, but not during isometric force generation, and implicate enhanced cross-bridge cycling in the cTnI-related pathway(s) to hypertrophy.

Deficient APC-cofactor activity of protein S Heerlen in degradation of factor Va Leiden: a possible mechanism of synergism between thrombophilic risk factors

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 523-531 ◽  
Author(s):  
Tusar Kanti Giri ◽  
Tomio Yamazaki ◽  
Núria Sala ◽  
Björn Dahlbäck ◽  
Pablo Garcı́a de Frutos
Keyword(s):  
Recombinant Protein ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor V ◽  
Wild Type ◽  
Genetic Traits ◽  
Factor Va ◽  
Type Protein ◽  
Wild Type Protein

Abstract In protein S Heerlen, an S-to-P (single-letter amino acid codes) mutation at position 460 results in the loss of glycosylation of N458. This polymorphism has been found to be slightly more prevalent in thrombophilic populations than in normal controls, particularly in cohorts of patients having free protein S deficiency. This suggests that carriers of the Heerlen allele may have an increased risk of thrombosis. We have now characterized the expression in cell cultures of recombinant protein S Heerlen and investigated the anticoagulant functions of the purified recombinant protein in vitro. Protein S Heerlen was synthesized and secreted equally well as wild-type protein S by transiently transfected COS-1 cells. The recombinant protein S Heerlen interacted with conformation-dependent monoclonal antibodies and bound C4b-binding protein to the same extent as wild-type protein S. Protein S Heerlen displayed reduced anticoagulant activity as cofactor to activated protein C (APC) in plasma-based assays, as well as in a factor VIIIa–degradation system. In contrast, protein S Heerlen functioned equally well as an APC cofactor in the degradation of factor Va as wild-type protein S did. However, when recombinant activated factor V Leiden (FVa:Q506) was used as APC substrate, protein S Heerlen was found to be a poor APC cofactor as compared with wild-type protein S. These in vitro results suggest a possible mechanism of synergy between protein S Heerlen and factor V Leiden that might be involved in the pathogenesis of thrombosis in individuals carrying both genetic traits.

scholarly journals Identification and Analysis of a Hyperactive Mutant Form of Drosophila P-Element Transposase

Genetics ◽  
2002 ◽  
Vol 162 (1) ◽  
pp. 217-227 ◽  
Author(s):  
Eileen L Beall ◽  
Matthew B Mahoney ◽  
Donald C Rio
Keyword(s):  
Dna Damage ◽  
Dna Cleavage ◽  
P Element ◽  
In Vitro Assays ◽  
Mutant Form ◽  
Wild Type ◽  
Type Protein ◽  
Wild Type Protein

Abstract Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

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