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At least 325 records · Page 18Linked to original sources
PubMed1998

Characterization of lethal Drosophila melanogaster alpha-actinin mutants.

We have partially characterized four Drosophila melanogaster alpha-actinin gene mutants, I(1)2Cb1, I(1)2Cb2, I(1)2Cb4, and I(1)2Cb5. We demonstrate that in each case the mutation is caused by a chromosomal rearrangement that precludes normal protein synthesis. In the absence of alpha-actinin, flies complete embryogenesis and develop into flaccid larvae that die within approximately 24 hr. These larvae have noticeable muscle dysfunction at hatching, although they, nevertheless, are capable of escaping from the egg membranes and of subsequent crawling movements. During larval development muscles degenerate, progressively limiting mobility and ultimately causing death. Electron microscopy of mutant muscle fibers reveals that myofibrils are grossly disrupted in one day old larvae and that electron-dense structures reminiscent of those seen in human nemaline myopathies are present throughout larval life. Our work rigorously demonstrates that alpha-actinin deficiencies are the cause of I(1)2Cb muscle defects. We anticipate that the alpha-actinin mutants described herein will facilitate in vivo tests of spectrin superfamily protein domain functions using a combination of directed mutagenesis and germline transformation.

Actinin↗
PubMed1992

Perturbations of Drosophila alpha-actinin cause muscle paralysis, weakness, and atrophy but do not confer obvious nonmuscle phenotypes.

We have investigated accumulation of alpha-actinin, the principal cross-linker of actin filaments, in four Drosophila fliA mutants. A single gene is variably spliced to generate one nonmuscle and two muscle isoforms whose primary sequence differences are confined to a peptide spanning the actin binding domain and first central repeat. In fliA3 the synthesis of an adult muscle-specific isoform is blocked in flight and leg muscles, while in fliA4 the synthesis of nonmuscle and both muscle-specific isoforms is severely reduced. Affected muscles are weak or paralyzed, and, in the case of fliA3, atrophic. Their myofibrils, while structurally irregular, are remarkably normal considering that they are nearly devoid of a major contractile protein. Also surprising is that no obvious nonmuscle cell abnormalities can be discerned despite the fact that both the fliA1- and fliA4-associated mutations perturb the nonmuscle isoform. Our observations suggest that alpha-actinin stabilizes and anchors thin filament arrays, rather than orchestrating their assembly, and further imply that alpha-actinin function is redundant in both muscle and nonmuscle cells.

Actin Cytoskeleton↗
PubMed1998

[Genomic studies of hereditary cardiomyopathies].

Cardiomyopathies (CMP) clinically and genetically belong to the heterogeneous group of myocardial diseases. Among them, three major clinical forms (hypertrophic, dilated, and restricted) are distinguished. Genetic factors play a substantial role in the etiology of dilated and hypertrophic CMP; family cases constitute more than 20% of these forms. Most familial cases of CMP are inherited as an autosomal dominant character. Autosomal recessive and X-linked forms are rare. Genetic basis for rare familial forms of restricted CMP is unclear. There are forms with strict maternal inheritance, which suggests the involvement of the mitochondrial genome. The nature of several CMP forms was determined and a number of genetic loci for this disease was revealed by modern methods of genetic mapping. In familial hypertrophic cardiomyopathy (FHC), four genes have been identified (those of beta-myosin heavy chain, alpha-tropomyosin, cardiac troponin T, and myosin-binding protein C), all of which encode sarcomeric proteins. Maternally inherited forms of FHC are associated with mutations in the mitochondrial tRNA genes. Linkage analysis in familial dilated CMP revealed at least five genetic loci on chromosomes 1, 3, 9, and X. X-linked forms of dilated CMP are caused by mutations in dystrophin gene, but the nature of autosomal forms is unclear. A recently recognized form of dilated CMP, arrhythmogenic CMP/right ventricular dysplasia (ARVD) is linked to two actinin gene loci on chromosomes 1 and 14. Genomic studies of CMP provided a basis for a new stage of "genetic cardiology", genetic mapping, which at present includes the quest of candidate genes for many other human cardiovascular diseases.

Cardiomegaly↗
PubMed2004

alpha-Tropomyosin mutations Asp(175)Asn and Glu(180)Gly affect cardiac function in transgenic rats in different ways.

To study the mechanisms by which missense mutations in alpha-tropomyosin cause familial hypertrophic cardiomyopathy, we generated transgenic rats overexpressing alpha-tropomyosin with one of two disease-causing mutations, Asp(175)Asn or Glu(180)Gly, and analyzed phenotypic changes at molecular, morphological, and physiological levels. The transgenic proteins were stably integrated into the sarcomere, as shown by immunohistochemistry using a human-specific anti-alpha-tropomyosin antibody, ARG1. In transgenic rats with either alpha-tropomyosin mutation, molecular markers of cardiac hypertrophy were induced. Ca(2+) sensitivity of cardiac skinned-fiber preparations from animals with mutation Asp(175)Asn, but not Glu(180)Gly, was decreased. Furthermore, elevated frequency and amplitude of spontaneous Ca(2+) waves were detected only in cardiomyocytes from animals with mutation Asp(175)Asn, suggesting an increase in intracellular Ca(2+) concentration compensating for the reduced Ca(2+) sensitivity of isometric force generation. Accordingly, in Langendorff-perfused heart preparations, myocardial contraction and relaxation were accelerated in animals with mutation Asp(175)Asn. The results allow us to propose a hypothesis of the pathogenetic changes caused by alpha-tropomyosin mutation Asp(175)Asn in familial hypertrophic cardiomyopathy on the basis of changes in Ca(2+) handling as a sensitive mechanism to compensate for alterations in sarcomeric structure.

Animals↗
PubMed2004

Cardiac myosin binding protein C: its role in physiology and disease.

Myosin binding protein-C (MyBP-C) is a thick filament-associated protein localized to the crossbridge-containing C zones of striated muscle sarcomeres. The cardiac isoform is composed of eight immunoglobulin I-like domains and three fibronectin 3-like domains and is known to be a physiological substrate of cAMP-dependent protein kinase. MyBP-C contributes to thick filament structure via interactions at its C-terminus with the light meromyosin section of the myosin rod and with titin. The protein also has a role in the regulation of contraction, due to the binding of its N-terminus to the subfragment-2 portion of myosin, which reduces actomyosin ATPase activity; phosphorylation abolishes this interaction, resulting in release of the "brake" on crossbridge cycling. Several structural models of the interaction of MyBP-C with myosin have been proposed, although its precise arrangement on the thick filament remains to be elucidated. Mutations in the gene encoding cardiac MyBP-C are a common cause of hypertrophic cardiomyopathy, and this has led to increased interest in the protein's function. Investigation of disease-causing mutations in domains with unknown function has led to further insights into the mechanism of cMyBP-C action. This Review aims to collate the published data on those aspects of MyBP-C that are well characterized and to consider new and emerging data that further define its structural and regulatory roles and its arrangement in the sarcomere. We also speculate on the mechanisms by which hypertrophic cardiomyopathy-causing truncation and missense mutations affect the normal functioning of the sarcomere.

Cardiomyopathy, Hypertrophic↗
PubMed2002

Ca(2+)-desensitizing effect of a deletion mutation Delta K210 in cardiac troponin T that causes familial dilated cardiomyopathy.

A deletion mutation Delta K210 in cardiac troponin T (cTnT) was recently found to cause familial dilated cardiomyopathy (DCM). To explore the effect of this mutation on cardiac muscle contraction under physiological conditions, we determined the Ca(2+)-activated force generation in permeabilized rabbit cardiac muscle fibers into which the mutant and wild-type cTnTs were incorporated by using our TnT exchange technique. The free Ca(2+) concentrations required for the force generation were higher in the mutant cTnT-exchanged fibers than in the wild-type cTnT-exchanged ones, with no statistically significant differences in maximal force-generating capability and cooperativity. Exchanging the mutant cTnT into isolated cardiac myofibrils also increased the free Ca(2+) concentrations required for the activation of ATPase. In contrast, a deletion mutation Delta E160 in cTnT that causes familial hypertrophic cardiomyopathy (HCM) decreased the free Ca(2+) concentrations required for force generation, just as in the case of the other HCM-causing mutations in cTnT. The results indicate that cTnT mutations found in the two distinct forms of cardiomyopathy (i.e., HCM and DCM) change the Ca(2+) sensitivity of cardiac muscle contraction in opposite directions. The present study strongly suggests that Ca(2+) desensitization of force generation in sarcomere is a primary mechanism for the pathogenesis of DCM associated with the deletion mutation Delta K210 in cTnT.

Adenosine Triphosphatases↗
PubMed1998

A dysfunctional desmin mutation in a patient with severe generalized myopathy.

Mice lacking desmin produce muscle fibers with Z disks and normal sarcomeric organization. However, the muscles are mechanically fragile and degenerate upon repeated contractions. We report here a human patient with severe generalized myopathy and aberrant intrasarcoplasmic accumulation of desmin intermediate filaments. Muscle tissue from this patient lacks the wild-type desmin allele and has a desmin gene mutation encoding a 7-aa deletion within the coiled-coil segment of the protein. We show that recombinant desmin harboring this deletion cannot form proper desmin intermediate filament networks in cultured cells, nor is it able to assemble into 10-nm filaments in vitro. These findings provide direct evidence that a mutation in desmin can cause human myopathies.

Amino Acid Sequence↗
PubMed1997

Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy.

Cardiac myosin binding protein C (MyBP-C) is a sarcomeric protein belonging to the intracellular immunoglobulin superfamily. Its function is uncertain, but for a decade evidence has existed for both structural and regulatory roles. The gene encoding cardiac MyBP-C (MYBPC3) in humans is located on chromosome 11p11.2, and mutations have been identified in this gene in unrelated families with familial hypertrophic cardiomyopathy (FHC). Detailed characterization of the MYBPC3 gene is essential for studies on gene regulation, analysis of the role of MyBP-C in cardiac contraction through the use of recombinant DNA technology, and mutational analyses of FHC. The organization of human MYBPC3 and screening for mutations in a panel of French families with FHC were established using polymerase chain reaction, single-strand conformation polymorphism, and sequencing. The MYBPC3 gene comprises > 21,000 base pairs and contains 35 exons. Two exons are unusually small in size, 3 bp each. We found six new mutations associated with FHC in seven unrelated French families. Four of these mutations are predicted to produce truncated cardiac MyBP-C polypeptides. The two others should each produce two aberrant proteins, one truncated and one mutated. The present study provides the first organization and sequence for an MyBP-C gene. The mutations reported here and previously in MYBPC3 result in aberrant transcripts that are predicted to encode significantly truncated cardiac MyBP-C polypeptides. This spectrum of mutations differs from the ones previously observed in other disease genes causing FHC. Our data strengthen the functional importance of MyBP-C in the regulation of cardiac work and provide the basis for further studies.

Base Sequence↗
PubMed2000

Requirements of Kettin, a giant muscle protein highly conserved in overall structure in evolution, for normal muscle function, viability, and flight activity of Drosophila.

Kettin is a giant muscle protein originally identified in insect flight muscle Z-discs. Here, we determined the entire nucleotide sequence of Drosophila melanogaster kettin, deduced the amino acid sequence of its protein product (540 kD) along with that of the Caenorhabditis elegans counterpart, and found that the overall primary structure of Kettin has been highly conserved in evolution. The main body of Drosophila Kettin consists of 35 immunoglobulin C2 domains separated by spacers. The central two thirds of spacers are constant in length and share in common two conserved motifs, putative actin binding sites. Neither fibronectin type III nor kinase domains were found. Kettin is present at the Z-disc in several muscle types. Genetic analysis showed that kettin is essential for the formation and maintenance of normal sarcomere structure of muscles and muscle tendons. Accordingly, embryos lacking kettin activity cannot hatch nor can adult flies heterozygous for the kettin mutation fly.

Amino Acid Sequence↗
PubMed2006

Calpain 3: a key regulator of the sarcomere?

Calpain 3 is a 94-kDa calcium-dependent cysteine protease mainly expressed in skeletal muscle. In this tissue, it localizes at several regions of the sarcomere through binding to the giant protein, titin. Loss-of-function mutations in the calpain 3 gene have been associated with limb-girdle muscular dystrophy type 2A (LGMD2A), a common form of muscular dystrophy found world wide. Recently, significant progress has been made in understanding the mode of regulation and the possible function of calpain 3 in muscle. It is now well accepted that it has an unusual zymogenic activation and that cytoskeletal proteins are one class of its substrates. Through the absence of cleavage of these substrates, calpain 3 deficiency leads to abnormal sarcomeres, impairment of muscle contractile capacity, and death of the muscle fibers. These data indicate a role for calpain 3 as a chef d'orchestre in sarcomere remodeling and suggest a new category of LGMD2 pathological mechanisms.

Animals↗
PubMed1995

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Amino Acid Sequence↗
PubMed2005

Is it time to include ion channel diseases among cardiomyopathies?

Heart muscle diseases are traditionally classified according to their peculiar pathophysiologic features such as "dilated," "hypertrophic," "restrictive," and "arrhythmogenic right ventricular" cardiomyopathy. The extraordinary advances accomplished in the last two decades in molecular genetics have allowed the identification of the genetic background of most of these conditions. According to the 1995 World Health Organization definition of cardiomyopathies as "diseases of the myocardium associated with cardiac dysfunction," they should include not only forms with hemodynamic dysfunction, but also conduction and rhythm disturbances. Arrhythmias are per se a sign of cardiac dysfunction and may reflect an underlying myocardial electrical disease with or without structural abnormalities as features. Nonstructural arrhythmogenic heart diseases include long and short QT syndromes, Brugada syndrome, Lènegre disease, and catecholaminergic polymorphic ventricular tachycardia. These conditions are defined as "channelopathies" because they are the consequence of cardiac ion channel gene mutations. Long and short QT syndromes are mostly caused by either sodium or potassium ion channel gene mutations; Brugada syndrome and Lènegre disease are both related to a defective sodium channel gene; and polymorphic ventricular tachycardia is the result of an abnormal ryanodine receptor regulating calcium release from the sarcoplasmic reticulum. These nonstructural inherited arrhythmic conditions should be regarded as cardiomyopathies because the myocyte is abnormal, although the heart is apparently intact. It is time for a new classification of cardiomyopathies taking into account the underlying gene mutations and the cellular level of expression of encoded proteins, thus distinguishing cytoskeleton (cytoskeletalopathies), desmosomal (desmosomalopathies), sarcomeric (sarcomyopathies), and ion channel (channelopathies) cardiomyopathies.

Arrhythmias, Cardiac↗
PubMed2004

Sorting of a nonmuscle tropomyosin to a novel cytoskeletal compartment in skeletal muscle results in muscular dystrophy.

Tropomyosin (Tm) is a key component of the actin cytoskeleton and >40 isoforms have been described in mammals. In addition to the isoforms in the sarcomere, we now report the existence of two nonsarcomeric (NS) isoforms in skeletal muscle. These isoforms are excluded from the thin filament of the sarcomere and are localized to a novel Z-line adjacent structure. Immunostained cross sections indicate that one Tm defines a Z-line adjacent structure common to all myofibers, whereas the second Tm defines a spatially distinct structure unique to muscles that undergo chronic or repetitive contractions. When a Tm (Tm3) that is normally absent from muscle was expressed in mice it became associated with the Z-line adjacent structure. These mice display a muscular dystrophy and ragged-red fiber phenotype, suggestive of disruption of the membrane-associated cytoskeletal network. Our findings raise the possibility that mutations in these tropomyosin and these structures may underpin these types of myopathies.

Animals↗
PubMed2005

Cardiac myofibril formation is not affected by modification of both N- and C-termini of sarcomeric tropomyosin.

Although the role of tropomyosin is well-defined in striated muscle, the precise mechanism of how tropomyosin functions is still unclear. It has been shown that extension of either N- or C-terminal ends of sarcomeric tropomyosin do not affect cardiac myofibrillogenesis, but it is not known whether simultaneous extension of both ends affects the process. For studying structural/functional relationships of sarcomeric tropomyosin, we have chosen the Ambystoma mexicanum because cardiac mutant hearts are deficient in sarcomeric tropomyosin. In this study, we have made an expression construct, pEGFP.TPM4alpha.E-L-FLAG, that, on transfection into normal and mutant axolotl hearts in organ culture, expresses GFP.TPM4alpha.E-L-FLAG fusion protein in which both the N- and C-termini of TPM4alpha are being extended. TPM4alpha is one of the three tropomyosins expressed in normal axolotl hearts. Both confocal and electron microscopic analyses show that this modified sarcomeric tropomyosin can form organized myofibrils in axolotl hearts.

Ambystoma mexicanum↗
PubMed1998

Identification of a new missense mutation in MyBP-C associated with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is a primary cardiac disease, characterised by idiopathic myocardial hypertrophy, and is caused by defects in sarcomeric protein encoding genes. One of these genes is cardiac myosin binding protein C (MyBP-C), in which a number of splice site and duplication mutations causing HCM have been described. During mutation screening of a South African HCM population by PCR-SSCP, a missense mutation, Arg654His, was detected in one proband. Although the mutation was present in his three adult children, only the proband himself was markedly affected. This is the first report of a disease associated missense mutation in MyBP-C which does not affect the myosin or titin binding domains.

Adult↗
PubMed2003

Biomolecular interactions between human recombinant beta-MyHC and cMyBP-Cs implicated in familial hypertrophic cardiomyopathy.

OBJECTIVE: Cardiac myosin-binding protein C (cMyBP-C) is a component of sarcomere that contains at least three putative myosin-binding sites. Mutations in its gene are implicated in familial hypertrophic cardiomyopathy (FHC) and most of them are predicted to produce C-terminal truncated cMyBP-Cs. The aim of the present study was to analyze whether cMyBP-C truncated mutants resulting from FHC mutations interact in vitro with human beta-MyHC. METHODS: Recombinant proteins were produced using the baculovirus/insect cell system, and wild type and three truncated cMyBP-Cs were purified using metal affinity chromatography. The interaction between recombinant proteins was analyzed in real time using biosensor technology on immobilized anti-beta-MyHC antibodies. RESULTS: Biomolecular interaction with beta-MyHC was detected for both wild type cMyBP-C and a truncated mutant lacking half of the C-terminal C10 domain. In contrast, no interaction with beta-MyHC was found for two truncated cMyBP-Cs lacking at least the C5-C9 region. CONCLUSIONS: Biosensor technology allows in vitro analysis of the interaction between human beta-MyHC and cMyBP-C mutants resulting from FHC mutations. The data show that the interaction depends on the size of the truncation. This suggests that, in the context of FHC, impairment of suitable interaction between beta-MyHC and some of the truncated cMyBP-Cs may promote degradation of the truncated proteins and therefore contribute to the development of the disease.

Animals↗
PubMed2002

Familial dilated cardiomyopathy.

Dilated cardiomyopathy is a disorder affecting heart muscle, characterized by ventricular dilation and reduced systolic function. It represents the most common cause of heart failure. Until recently, dilated cardiomyopathy was considered an exclusively sporadic and idiopathic disease. Now, as defined by the World Health Organization, cardiomyopathy includes not only the idiopathic form, but secondary ones such as ischemic or hypertensive. It is estimated that familial occurrence accounts for 30% of cases of idiopathic dilated cardiomyopathy. The most common mode of inheritance is the autosomal dominant type. The X-linked, autosomal recessive and mitochondrial forms are less common. Different genes or loci are responsible for the cardiac dilatation, and code for sarcomeric, cytoskeleton and nuclear lamina proteins. The molecular interactions of the mutated proteins with factors such as infectious agents or alcohol could explain the variety of presenting signs and symptoms of this type of cardiomyopathy. Recently the European Society of Cardiology published a definition and a protocol for the study of familial dilated cardiomyopathies. Genetic research in the field of dilated cardiomyopathy can increase our understanding of its pathogenesis and lead to new treatment modalities for the disease.

Cardiomyopathy, Dilated↗