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Hugh Watkins - One of the best experts on this subject based on the ideXlab platform.
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familial dilated cardiomyopathy caused by an Alpha Tropomyosin mutation the distinctive natural history of sarcomeric dcm
Journal of Cardiac Failure, 2009Co-Authors: Neal K Lakdawala, Hugh Watkins, Paul Robinson, Lisa Dellefave, Elizabeth Sparks, Allison L Cirino, Steve Depalma, Birgit H Funke, Steven D Colan, C RedwoodAbstract:Objectives To further define the role of sarcomere mutations in DCM and associated clinical phenotypes.
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the effect of mutations in Alpha Tropomyosin e40k and e54k that cause familial dilated cardiomyopathy on the regulatory mechanism of cardiac muscle thin filaments
Journal of Biological Chemistry, 2007Co-Authors: M Mirza, Hugh Watkins, Paul Robinson, Charles Redwood, Elena Kremneva, Oneal Copeland, Olga P Nikolaeva, Dimitry Levitsky, Mohammed ElmezgueldiAbstract:Abstract E40K and E54K mutations in α-Tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser α-Tropomyosin (AS-α-Tm) expressed in Escherichia coli, that both mutations decrease Ca2+ sensitivity. E40K also reduces Vmax of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-α-Tm, and the two dilated cardiomyopathy-causing mutants. AS-α-Tm has a lower cooperative unit size (6.5) than native α-Tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the KT value was the same for all actin-Tm species; however, the KT value of actin-Tm-troponin at pCa 5 was 50% less for AS-α-Tm E40K than for AS-α-Tm and AS-α-Tm E54K. Kb, the “closed” to “blocked” equilibrium constant, was the same for all Tropomyosin species. The E40K mutation reduced the affinity of Tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-α-Tm showed that domain 3, assigned to the N terminus of Tropomyosin, was strongly destabilized by both mutations. Additionally with AS-α-Tm E54K, we observed a unique new domain at 55 °C accounting for 25% of enthalpy indicating stabilization of part of the Tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on Tropomyosin structure and function.
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effect of hypertrophic cardiomyopathy mutations in human cardiac muscle Alpha Tropomyosin asp175asn and glu180gly on the regulatory properties of human cardiac troponin determined by in vitro motility assay
Journal of Molecular and Cellular Cardiology, 2000Co-Authors: Wu Bing, Hugh Watkins, A Knott, Charles Redwood, Giovanna Esposito, Ian Purcell, Steven B MarstonAbstract:The properties of mutant contractile proteins that cause hypertrophic cardiomyopathy (HCM) have been investigated in expression studies and in mouse models. There is growing evidence that the precise isoforms of both the mutated protein and its interacting partners can qualitatively influence the effects of the mutation. We therefore investigated the functional effects of two HCM mutations in Alpha -Tropomyosin, Asp175Asn and Glu180Gly, in the in vitro motility assay using recombinant human Alpha -Tropomyosin, expressed with an N-terminal alanine-serine extension (AStm) to mimic acetylation in vivo, and purified native human cardiac troponin. The expected switching off of reconstituted filament movement at pCa9, and switching on at pCa5, was observed with no difference in fraction of filaments motile or filament velocity, between wild-type and mutant filaments. However, we observed increased Ca(2+)sensitivity of fraction of filaments motile using the mutant Tropomyosin compared to wild-type (DeltaEC(50)+0.082+/-0. 019 pCa units for Asp175Asn and +0.115+/-0.021 for Glu180Gly). Indirect measurements using immobilized Alpha -actinin to retard filament movement showed that filaments reconstituted with mutant AStm produced the same force as wild-type filaments. The results using human cardiac regulatory proteins reveal different effects of the HCM mutations in Tropomyosin compared to studies using heterologous systems. By performing parallel experiments using either human cardiac or rabbit skeletal troponin we show that the cardiac-specific phenotype of HCM mutations in Alpha -Tropomyosin is not the result of more marked functional changes when interacting with cardiac troponin.
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effects of two hypertrophic cardiomyopathy mutations in Alpha Tropomyosin asp175asn and glu180gly on ca2 regulation of thin filament motility
Biochemical and Biophysical Research Communications, 1997Co-Authors: Wu Bing, Hugh Watkins, C Redwood, Giovanna Esposito, Ian Purcell, Steven B MarstonAbstract:Abstract The functional properties of wild type α-Tropomyosin expressed in E. coli with an alanine-serine N-terminal leader (AS-α-Tm) were compared with those of AS-α-Tm with either of two missense mutations (Asp175Asn and Glu180Gly) shown to cause familial hypertrophic cardiomyopathy (FHC). Wild type AS-α-Tm and AS-α-Tm(Asp175Asn) binding to actin was indistinguishable from rabbit skeletal muscle ab-Tropomyosin whilst the affinity of AS-α-Tm (Glu180Gly) was about threefold weaker. In vitro motility assays were performed with AS-α-Tropomyosin incorporated into skeletal muscle actin-rhodamine phalloidin filaments moving over skeletal muscle heavy meromyosin. Under relaxing conditions (pCa9), troponin added to actin filaments containing AS-α-Tropomyosin or mutant Tropomyosins resulted in normal switch-off, with a decrease in the fraction filaments moving from >80% to 2+ - regulation of thin filaments, presumably via altered interaction with troponin.
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clinical features of hypertrophic cardiomyopathy caused by mutation of a hot spot in the Alpha Tropomyosin gene
Journal of the American College of Cardiology, 1997Co-Authors: Domenico A Coviello, Hans-peter Vosberg, Hugh Watkins, Barry J Maron, L Thierfelder, Paolo Spirito, Frederick J Schoen, J G SeidmanAbstract:Abstract Objectives. We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the Alpha-Tropomyosin gene in affected subjects from three unrelated families. Background. Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by Alpha-Tropomyosin mutations have been hampered by the rarity of these genetic defects. Methods. The haplotypes of three kindreds with FHC due to an Alpha-Tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared. Results. Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness ± SD: 24 ± 4.5 mm in anterior septum of Family DT; 15 ± 2.7 mm in anterior septum and free wall of Family DB; 18 ± 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable. Conclusions. Nucleotide residue 579 in the Alpha-Tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the Alpha-Tropomyosin mutation Asp175Asn. (J Am Coll Cardiol 1997;29:635–40)
J G Seidman - One of the best experts on this subject based on the ideXlab platform.
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effects of two familial hypertrophic cardiomyopathy causing mutations on Alpha Tropomyosin structure and function
Biochemistry, 1997Co-Authors: Nina L Golitsina, Norma J. Greenfield, L Thierfelder, J G Seidman, Kenji Iizuka, Christine E Seidman, Sherwin S Lehrer, Sarah E HitchcockdegregoriAbstract:Missense mutations in Alpha-Tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human Tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of Tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of Tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled Tropomyosins differed in their response to myosin S1 binding to the actin-Tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant Tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.
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clinical features of hypertrophic cardiomyopathy caused by mutation of a hot spot in the Alpha Tropomyosin gene
Journal of the American College of Cardiology, 1997Co-Authors: Domenico A Coviello, Hans-peter Vosberg, Hugh Watkins, Barry J Maron, L Thierfelder, Paolo Spirito, Frederick J Schoen, J G SeidmanAbstract:Abstract Objectives. We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the Alpha-Tropomyosin gene in affected subjects from three unrelated families. Background. Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by Alpha-Tropomyosin mutations have been hampered by the rarity of these genetic defects. Methods. The haplotypes of three kindreds with FHC due to an Alpha-Tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared. Results. Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness ± SD: 24 ± 4.5 mm in anterior septum of Family DT; 15 ± 2.7 mm in anterior septum and free wall of Family DB; 18 ± 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable. Conclusions. Nucleotide residue 579 in the Alpha-Tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the Alpha-Tropomyosin mutation Asp175Asn. (J Am Coll Cardiol 1997;29:635–40)
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mutations in the cardiac myosin binding protein c gene on chromosome 11 cause familial hypertrophic cardiomyopathy
Nature Genetics, 1995Co-Authors: David A Conner, John A. Jarcho, Barry J Maron, L Thierfelder, Calum A. Macrae, J G SeidmanAbstract:Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disorder manifesting as cardiac hypertrophy with myocyte disarray and an increased risk of sudden death. Mutations in five different loci cause FHC and 3 disease genes have been identified: beta cardiac myosin heavy chain, Alpha Tropomyosin and cardiac troponin T. Because these genes encode contractile proteins, other FHC loci are predicted also to encode sarcomere components. Two further FHC loci have been mapped to chromosomes 11p13-q13 (CMH4, ref. 6) and 7q3 (ref. 7). The gene encoding the cardiac isoform of myosin binding protein-C (cardiac MyBP-C) has recently been assigned to chromosome 11p11.2 and proposed as a candidate FHC gene. Cardiac MyBP-C is arrayed transversely in sarcomere A-bands and binds myosin heavy chain in thick filaments and titin in elastic filaments. Phosphorylation of MyBP-C appears to modulate contraction. We report that cardiac MyBP-C is genetically linked to CMH4 and demonstrate a splice donor mutation in one family with FHC and a duplication mutation in a second. Both mutations are predicted to disrupt the high affinity, C-terminal, myosin-binding domain of cardiac MyBP-C. These findings define cardiac MyBP-C mutations as the cause of FHC on chromosome 11p and reaffirm that FHC is a disease of the sarcomere.
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a de novo mutation in Alpha Tropomyosin that causes hypertrophic cardiomyopathy
Circulation, 1995Co-Authors: Hugh Watkins, J G Seidman, R Anan, Domenico A Coviello, Paolo Spirito, Christine E SeidmanAbstract:Background Two missense mutations in the gene for α-Tropomyosin have been described that segregate with hypertrophic cardiomyopathy in single families. To confirm that these mutations are the cause of the disease, we have investigated the origins of one of these mutations, Asp175Asn, in a third and unrelated family. Methods and Results The presence or absence of an α-Tropomyosin mutation and the haplotypes of the flanking chromosomal regions were determined for members of a family with hypertrophic cardiomyopathy. Haplotypes were constructed by use of an intragenic polymorphism and 10 flanking polymorphisms spanning a region of 35 centimorgans. The Asp175Asn missense mutation was present in the proband and his two affected offspring but not in any of the proband’s three siblings. Although both parents were deceased, the haplotypes of the four parental chromosomes could be reconstructed. One parental chromosome was transmitted to two offspring: one bearing the Asp175Asn mutation (the affected proband) and ...
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mutations in the genes for cardiac troponin t and Alpha Tropomyosin in hypertrophic cardiomyopathy
The New England Journal of Medicine, 1995Co-Authors: Hugh Watkins, William J. Mckenna, L Thierfelder, H J Suk, R Anan, A Odonoghue, P Spirito, Akira Matsumori, C S Moravec, J G SeidmanAbstract:Background Familial hypertrophic cardiomyopathy can be caused by mutations in the genes for β cardiac myosin heavy chain, α-Tropomyosin, or cardiac troponin T. It is not known how often the disease is caused by mutations in the Tropomyosin and troponin genes, and the associated clinical phenotypes have not been carefully studied. Methods Linkage between polymorphisms of the α-Tropomyosin gene or the cardiac troponin T gene and hypertrophic cardiomyopathy was assessed in 27 families. In addition, 100 probands were screened for mutations in the α-Tropomyosin gene, and 26 were screened for mutations in the cardiac troponin T gene. Life expectancy, the incidence of sudden death, and the extent of left ventricular hypertrophy were compared in patients with different mutations. Results Genetic analyses identified only one α-Tropomyosin mutation, identical to one previously described. Five novel mutations in cardiac troponin were identified, as well as a further example of a previously described mutation. The cl...
Sarah E Hitchcockdegregori - One of the best experts on this subject based on the ideXlab platform.
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structure of the n terminus of a nonmuscle Alpha Tropomyosin in complex with the c terminus implications for actin binding
Biochemistry, 2009Co-Authors: Norma J. Greenfield, Lucy Kotlyanskaya, Sarah E HitchcockdegregoriAbstract:Tropomyosin is a coiled-coil actin binding protein that stabilizes the filament, protects it from severing, and cooperatively regulates actin’s interaction with myosin. Depending on the first coding exon, Tropomyosins are low molecular weight (LMW), found in the cytoskeleton and predominant in transformed cells, or high molecular weight (HMW), found in muscle and nonmuscle cells. The N- and C-terminal ends form a complex that allows Tropomyosin to associate N terminus-to-C terminus along the actin filament. We determined the structure of a LMW Tropomyosin N-terminal model peptide complexed with a smooth/nonmuscle Tropomyosin C-terminal peptide. Using NMR and circular dichroism we showed that both ends become more helical upon complex formation but that the C-terminal peptide is partially unfolded at 20 °C. The first five residues of the N terminus that are disordered in the free peptide are more helical and are part of the overlap complex. NMR data indicate residues 2−17 bind to the C terminus in the comp...
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the structure of the carboxyl terminus of striated Alpha Tropomyosin in solution reveals an unusual parallel arrangement of interacting Alpha helices
Biochemistry, 2003Co-Authors: Norma J. Greenfield, Gaetano T. Montelione, Thomas Palm, G V T Swapna, Yuanpeng Huang, Sarah Graboski, Sarah E HitchcockdegregoriAbstract:Coiled coils are well-known as oligomerization domains, but they are also important sites of protein-protein interactions. We determined the NMR solution structure and backbone 1 5 N relaxation rates of a disulfide cross-linked, two-chain, 37-residue polypeptide containing the 34 C-terminal residues of striated muscle α-Tropomyosin, TM9a 2 5 1 - 2 8 4 . The peptide binds to the N-terminal region of TM and to the Tropomyosin-binding domain of the regulatory protein, troponin T. Comparison of the NMR solution structure of TM9a 2 5 1 - 2 8 4 with the X-ray structure of a related peptide [Li, Y., Mui, S., Brown, J. H., Strand, J., Reshetnikova, L., Tobacman, L. S., and Cohen, C. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7378-7383] reveals significant differences. In solution, residues 253-269 (like most of the Tropomyosin molecule) form a canonical coiled coil. Residues 270-279, however, are parallel, linear helices, novel for Tropomyosin. The packing between the parallel helices results from unusual interface residues that are atypical for coiled coils. Y267 has poor packing at the coiled-coil interface and a lower R 2 relaxation rate than neighboring residues, suggesting there is conformational flexibility around this residue. The last five residues are nonhelical and flexible. The exposed surface presented by the parallel helices, and the flexibility around Y267 and the ends, may facilitate binding to troponin T and formation of complexes with the N-terminus of Tropomyosin and actin. We propose that unusual packing and flexibility are general features of coiled-coil domains in proteins that are involved in intermolecular interactions.
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solution nmr structure and folding dynamics of the n terminus of a rat non muscle Alpha Tropomyosin in an engineered chimeric protein
Journal of Molecular Biology, 2001Co-Authors: Norma J. Greenfield, Gaetano T. Montelione, Yuanpeng J Huang, Thomas Palm, G V T Swapna, Daniel Monleon, Sarah E HitchcockdegregoriAbstract:Tropomyosin is an Alpha-helical coiled-coil protein that aligns head-to-tail along the length of the actin filament and regulates its function. The solution structure of the functionally important N terminus of a short 247-residue non-muscle Tropomyosin was determined in an engineered chimeric protein, GlyTM1bZip, consisting of the first 19 residues of rat short Alpha-Tropomyosin and the last 18 residues of the GCN4 leucine zipper. A gene encoding GlyTM1bZip was synthesized, cloned and expressed in Escherichia coli. Triple resonance NMR spectra were analyzed with the program AutoAssign to assign its backbone resonances. Multidimensional nuclear Overhauser effect spectra, X-filtered spectra and (3)J(H(N)-H(Alpha)) scalar coupling were analyzed using AutoStructure. This is the first application of this new program to determine the three-dimensional structure of a symmetric homodimer and a structure not previously reported. Residues 7-35 in GlyTM1bZip form a coiled coil, but neither end is helical. Heteronuclear (15)N-(1)H nuclear Overhauser effect data showed that the non-helical N-terminal residues are flexible. The (13)C' chemical shifts of the coiled-coil backbone carbonyl groups in GlyTM1bZip showed a previously unreported periodicity, where resonances arising from residues at the coiled-coil interface in a and d positions of the heptad repeat were displaced relatively upfield and those arising from residues in c positions were displaced relatively downfield. Heteronuclear single quantum coherence spectra, collected as a function of temperature, showed that cross-peaks arising from the Alpha-helical backbone and side-chains at the coiled-coil interface broadened or shifted with T(M) values approximately 20 degrees C lower than the loss of Alpha-helix measured by circular dichroism, suggesting the presence of a folding intermediate. The side-chain of Ile14, a residue essential for binding interactions, exhibited multiple conformations. The conformational flexibility of the N termini of short Tropomyosins may be important for their binding specificity.
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effects of two familial hypertrophic cardiomyopathy causing mutations on Alpha Tropomyosin structure and function
Biochemistry, 1997Co-Authors: Nina L Golitsina, Norma J. Greenfield, L Thierfelder, J G Seidman, Kenji Iizuka, Christine E Seidman, Sherwin S Lehrer, Sarah E HitchcockdegregoriAbstract:Missense mutations in Alpha-Tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human Tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of Tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of Tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled Tropomyosins differed in their response to myosin S1 binding to the actin-Tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant Tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.
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requirement of amino terminal modification for striated muscle Alpha Tropomyosin function
Journal of Biological Chemistry, 1994Co-Authors: M Urbancikova, Sarah E HitchcockdegregoriAbstract:Striated muscle Alpha-Tropomyosin expressed in Escherichia coli is unacetylated, polymerizes poorly, and binds weakly to F-actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). To define the structural requirements of NH2-terminal modification for striated Tropomyosin function, an acetylated recombinant Tropomyosin and an unacetylated short fusion recombinant Tropomyosin were compared. An acetylated recombinant chicken striated muscle Alpha-Tropomyosin was expressed in insect Sf9 cells using the baculovirus expression vector system. The purified Tropomyosin (approximately 15 mg/liter of insect cell suspension) polymerized, comigrated with chicken striated Alpha-Tropomyosin purified from muscle on two-dimensional polyacrylamide gels, was blocked at the NH2 terminus, and had the same actin affinity as muscle Tropomyosin. These results conclusively show the importance of NH2-terminal acetylation for striated Tropomyosin function. To learn if a short fusion peptide would substitute for amino-terminal acetylation, Tropomyosin with AlaSer-Arg on the NH2 terminus was constructed and expressed in E. coli as an unacetylated protein. This f3-Tropomyosin bound to actin with a 10-fold higher affinity than striated muscle Alpha-TM and, unlike muscle Tropomyosin, exhibited a shear-dependent viscosity. The altered function of f3-Tropomyosin shows that the naturally occurring acetylated NH2 terminus is required for full, normal function. It is proposed that a major requirement for cooperative binding of striated muscle Tropomyosin to actin is modification of the Alpha-amino group of methionine to be an amide, as when acetylated or in a peptide bond in a fusion protein, to make the extreme NH2 terminus more hydrophobic. The results are discussed in terms of known coiled coil structure.
L Thierfelder - One of the best experts on this subject based on the ideXlab platform.
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autonomic cardiac control in animal models of cardiovascular diseases ii variability analysis in transgenic rats with Alpha Tropomyosin mutations asp175asn and glu180gly
Biomedizinische Technik, 2007Co-Authors: Dirk Wernicke, Niels Wessel, Hagen Malberg, Ralph Plehm, Robert Bauernschmitt, L ThierfelderAbstract:Animal models of cardiovascular diseases allow to investigate relevant pathogenetic mechanisms in detail. In the present study, the mutations Asp175Asn and Glu180Gly in Alpha-Tropomyosin (TPM1), known cause familiar hypertrophic cardiomyopathy (FHC) were studied for changes in hemodynamic parameters and spontaneous baroreflex regulation in transgenic rats in comparison to transgenic and non-transgenic controls by telemetry. Heart rate variability (HRV) and blood pressure variability (BPV) were analyzed using time- and frequency domain, as well as non-linear measures. The dual sequence method was used for the estimation of the baroreflex regulation. In transgenic rats harboring mutated TPM1, changes in HRV were detected during exercise, but not at rest. Both mutations, Asp175Asn and Glu180Gly, caused increased low frequency power. In addition, in animals with mutation Asp175Asn a reduced total HRV was observed. BPV did not show any differences between all transgenic animal lines. During exercise, a strong increase in the number of bradycardic and tachycardic fluctuations accompanied with decreased baroreflex sensitivity (BRS) was detected in animals with either TPM1 mutation, Asp175Asn or Glu180Gly. These data suggest, that the analysis of cardiac autonomic control, particularly of baroreflex regulation, represents a powerful non-invasive approach to investigate the effects of subtle changes in sarcomeric architecture on cardiac physiology in vivo. In case of mutations Asp175Asn or Glu180Gly in TPM1, early detection of alterations in autonomic cardiac control could help to prevent sudden cardiac death in affected persons.
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effects of two familial hypertrophic cardiomyopathy causing mutations on Alpha Tropomyosin structure and function
Biochemistry, 1997Co-Authors: Nina L Golitsina, Norma J. Greenfield, L Thierfelder, J G Seidman, Kenji Iizuka, Christine E Seidman, Sherwin S Lehrer, Sarah E HitchcockdegregoriAbstract:Missense mutations in Alpha-Tropomyosin can cause familial hypertrophic cardiomyopathy. The effects of two of these, Asp175Asn and Glu180Gly, have been tested on the structure and function of recombinant human Tropomyosin expressed in Escherichia coli. The F-actin affinity (measured by cosedimentation) of Glu180Gly was similar to that of wild-type, but Asp175Asn was more than 2-fold weaker, whether or not troponin was present. The mutations had no apparent effect on the affinity of Tropomyosin for troponin. The mutations had a small effect on the overall stability (measured using circular dichroism) but caused increased local flexibility or decreased local stability, as evaluated by the higher excimer/monomer ratios of Tropomyosin labeled with pyrene maleimide at Cys 190. The pyrene-labeled Tropomyosins differed in their response to myosin S1 binding to the actin-Tropomyosin filament. The conformations of the two mutants were different from each other and from wild-type in the myosin S1-induced on-state of the thin filament. Even though both mutant Tropomyosins bound cooperatively to actin, they did not respond with the same conformational change as wild-type when myosin S1 switched the thin filament from the off- to the on-state.
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clinical features of hypertrophic cardiomyopathy caused by mutation of a hot spot in the Alpha Tropomyosin gene
Journal of the American College of Cardiology, 1997Co-Authors: Domenico A Coviello, Hans-peter Vosberg, Hugh Watkins, Barry J Maron, L Thierfelder, Paolo Spirito, Frederick J Schoen, J G SeidmanAbstract:Abstract Objectives. We studied the clinical and genetic features of familial hypertrophic cardiomyopathy (FHC) caused by an Asp175Asn mutation in the Alpha-Tropomyosin gene in affected subjects from three unrelated families. Background. Correlation of genotype and phenotype has provided important information in FHC caused by beta-cardiac myosin and cardiac troponin T mutations. Comparable analyses of hypertrophic cardiomyopathy caused by Alpha-Tropomyosin mutations have been hampered by the rarity of these genetic defects. Methods. The haplotypes of three kindreds with FHC due to an Alpha-Tropomyosin gene mutation, Asp175Asn, were analyzed. The cardiac histopathologic findings of this mutation are reported. Distribution of left ventricular hypertrophy in affected members was assessed by two-dimensional echocardiography, and patient survival rates were compared. Results. Genetic studies defined unique haplotypes in the three families, demonstrating that independent mutations caused the disease in each. The Asp175Asn mutation caused cardiac histopathologic findings of myocyte hypertrophy, disarray and replacement fibrosis. The severity and distribution of left ventricular hypertrophy varied considerably in affected members from the three families (mean maximal wall thickness ± SD: 24 ± 4.5 mm in anterior septum of Family DT; 15 ± 2.7 mm in anterior septum and free wall of Family DB; 18 ± 2.1 mm in posterior septum of Family MI), but survival was comparable and favorable. Conclusions. Nucleotide residue 579 in the Alpha-Tropomyosin gene may have increased susceptibility to mutation. On cardiac histopathologic study, defects in this sarcomere thin filament component are indistinguishable from other genetic etiologies of hypertrophic cardiomyopathy. The Asp175Asn mutation can elicit different morphologic responses, suggesting that the hypertrophic phenotype is modulated not by genetic etiologic factors alone. In contrast, prognosis reflected genotype; near normal life expectancy is found in hypertrophic cardiomyopathy caused by the Alpha-Tropomyosin mutation Asp175Asn. (J Am Coll Cardiol 1997;29:635–40)
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mutations in the cardiac myosin binding protein c gene on chromosome 11 cause familial hypertrophic cardiomyopathy
Nature Genetics, 1995Co-Authors: David A Conner, John A. Jarcho, Barry J Maron, L Thierfelder, Calum A. Macrae, J G SeidmanAbstract:Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disorder manifesting as cardiac hypertrophy with myocyte disarray and an increased risk of sudden death. Mutations in five different loci cause FHC and 3 disease genes have been identified: beta cardiac myosin heavy chain, Alpha Tropomyosin and cardiac troponin T. Because these genes encode contractile proteins, other FHC loci are predicted also to encode sarcomere components. Two further FHC loci have been mapped to chromosomes 11p13-q13 (CMH4, ref. 6) and 7q3 (ref. 7). The gene encoding the cardiac isoform of myosin binding protein-C (cardiac MyBP-C) has recently been assigned to chromosome 11p11.2 and proposed as a candidate FHC gene. Cardiac MyBP-C is arrayed transversely in sarcomere A-bands and binds myosin heavy chain in thick filaments and titin in elastic filaments. Phosphorylation of MyBP-C appears to modulate contraction. We report that cardiac MyBP-C is genetically linked to CMH4 and demonstrate a splice donor mutation in one family with FHC and a duplication mutation in a second. Both mutations are predicted to disrupt the high affinity, C-terminal, myosin-binding domain of cardiac MyBP-C. These findings define cardiac MyBP-C mutations as the cause of FHC on chromosome 11p and reaffirm that FHC is a disease of the sarcomere.
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mutations in the genes for cardiac troponin t and Alpha Tropomyosin in hypertrophic cardiomyopathy
The New England Journal of Medicine, 1995Co-Authors: Hugh Watkins, William J. Mckenna, L Thierfelder, H J Suk, R Anan, A Odonoghue, P Spirito, Akira Matsumori, C S Moravec, J G SeidmanAbstract:Background Familial hypertrophic cardiomyopathy can be caused by mutations in the genes for β cardiac myosin heavy chain, α-Tropomyosin, or cardiac troponin T. It is not known how often the disease is caused by mutations in the Tropomyosin and troponin genes, and the associated clinical phenotypes have not been carefully studied. Methods Linkage between polymorphisms of the α-Tropomyosin gene or the cardiac troponin T gene and hypertrophic cardiomyopathy was assessed in 27 families. In addition, 100 probands were screened for mutations in the α-Tropomyosin gene, and 26 were screened for mutations in the cardiac troponin T gene. Life expectancy, the incidence of sudden death, and the extent of left ventricular hypertrophy were compared in patients with different mutations. Results Genetic analyses identified only one α-Tropomyosin mutation, identical to one previously described. Five novel mutations in cardiac troponin were identified, as well as a further example of a previously described mutation. The cl...
Nigel G. Laing - One of the best experts on this subject based on the ideXlab platform.
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de novo missense mutation in a constitutively expressed exon of the slow Alpha Tropomyosin gene tpm3 associated with an atypical sporadic case of nemaline myopathy
Neuromuscular Disorders, 2002Co-Authors: H J Durling, Peter Reilich, J Mullerhocker, B Mendel, D Pongratz, Carina Wallgrenpettersson, Peter W Gunning, Hanns Lochmuller, Nigel G. LaingAbstract:Abstract We describe an atypical case of nemaline myopathy with an unusual distribution of muscle weakness who presented at 14 years of age with kyphoscoliosis . In this patient, we demonstrate heterozygosity for a de novo CGT–CAT (Arg167His) mutation in a constitutively expressed exon (exon 5) of slow Alpha-Tropomyosin (TPM3). This is the first mutation identified in a constitutively expressed exon of TPM3 in a nemaline myopathy patient, but is similar to recently described mutations in beta-Tropomyosin (TPM2) associated with nemaline myopathy and mutations in fast Alpha-Tropomyosin (TPM1) which cause hypertrophic cardiomyopathy.
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nemaline myopathy a clinical study of 143 cases
Annals of Neurology, 2001Co-Authors: Nigel G. Laing, Monique M. Ryan, Susan T. Iannaccone, Alan H. Beggs, Corinne D. Strickland, Christina Schnell, Lloyd K. Shield, Graeme Morgan, Kathryn N. NorthAbstract:We report 143 Australian and North American cases of primary nemaline myopathy. As classified by the European Neuromuscular Centre guidelines, 23 patients had severe congenital, 29 intermediate congenital, 66 typical congenital, 19 childhood-onset, and 6 adult-onset nemaline myopathy. Inheritance was autosomal recessive in 29 patients, autosomal dominant in 41, sporadic in 72, and indeterminate in 1. Twenty-two patients had skeletal muscle actin mutations and 4 had mutations in the Alpha-Tropomyosin(slow) gene. Obstetric complications occurred in 49 cases. Seventy-five patients had significant respiratory disease during the first year of life, and 79 had feeding difficulties. Atypical features in a minority of cases included arthrogryposis, central nervous system involvement, and congenital fractures. Progressive distal weakness developed in a minority of patients. Thirty patients died, the majority during the first 12 months of life. All deaths were due to respiratory insufficiency, which was frequently underrecognized in older patients. Arthrogryposis, neonatal respiratory failure, and failure to achieve early motor milestones were associated with early mortality. Morbidity from respiratory tract infections and feeding difficulties frequently diminished with increasing age. Aggressive early management is warranted in most cases of congenital nemaline myopathy.
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homozygosity for a nonsense mutation in the Alpha Tropomyosin slow gene tpm3 in a patient with severe infantile nemaline myopathy
Neuromuscular Disorders, 1999Co-Authors: J Briner, Kathryn N. North, S D Wilton, Carina Wallgrenpettersson, E Boltshauser, M Davis, Nigel G. LaingAbstract:The nemaline myopathies are muscle disorders of variable severity and age of onset, with characteristic nemaline bodies in the sarcoplasm. Genes for dominant (NEM1) and recessive (NEM2A) nemaline myopathy have been localised to chromosomes one and two, respectively. A missense mutation in the Alpha-Tropomyosin gene (TPM3) has been associated with NEM1 in one family. Probands from 76 other nemaline myopathy families have now been screened for TPM3 mutations. One proband, who was not noted to have any weakness neonatally, but who died at 21 months of age, was shown to be homozygous for a single strand conformation polymorphism (SSCP) in skeletal-muscle-specific exon 1 of TPM3. Sequencing revealed homozygosity for a nonsense mutation at codon 31 (CAG to TAG). The patient should have no functioning Alpha-Tropomyosin slow protein. The nemaline bodies in this patient were exclusively in type one fibres, consistent with the expression of TPM3 only in type one fibres.
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assignment of the human Alpha Tropomyosin gene tpm4 to band 19p13 1 by fluorescence in situ hybridization
Cytogenetic and Genome Research, 1996Co-Authors: Steve D Wilton, H J Eyre, L Lim, S D Dorosz, H C Gunn, D F Callen, Nigel G. LaingAbstract:Sequence-tagged sites (STSs) were developed for the human α-Tropomyosin gene TPM4. One STS was used to amplify DNA from somatic cell hybrids to localize TPM4 to chromosome 19. The other, a product from a long-range PCR, was used directly as a probe to refine the localization of TPM4 to 19p13.1 by fluorescence in situ hybridization to metaphase chromosome spreads.
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a mutation in the Alpha Tropomyosin gene tpm3 associated with autosomal dominant nemaline myopathy
Nature Genetics, 1995Co-Authors: Nigel G. Laing, S D Wilton, P A Akkari, S Dorosz, K Boundy, C Kneebone, Peter C Blumbergs, S White, Hugh WatkinsAbstract:Nemaline myopathies are diseases characterized by the presence in muscle fibres of pathognomonic rod bodies. These are composed largely of Alpha−actinin and actin. We have identified a missense mutation in the Alpha−Tropomyosin gene, TPM3, which segregates completely with the disease in a family whose autosomal dominant nemaline myopathy we had previously localized to chromosome 1p13−q25. The mutation substitutes an arginine residue for a highly conserved methionine in a putative actin−binding site near the N terminus of the Alpha−Tropomyosin. The mutation may strengthen Tropomyosin − actin binding, leading to rod body formation, by adding a further basic residue to the postulated actin−binding motif.