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PubMed2005

Role of the serum response factor in regulating contractile apparatus gene expression and sarcomeric integrity in cardiomyocytes.

The serum response factor (SRF) is a transcriptional regulator required for mesodermal development, including heart formation and function. Previous studies have described the role of SRF in controlling expression of structural genes involved in conferring the myogenic phenotype. Recent studies by us and others have demonstrated embryonic lethal cardiovascular phenotypes in SRF-null animals, but have not directly addressed the mechanistic role of SRF in controlling broad regulatory programs in cardiac cells. In this study, we used a loss-of-function approach to delineate the role of SRF in cardiomyocyte gene expression and function. In SRF-null neonatal cardiomyocytes, we observed severe defects in the contractile apparatus, including Z-disc and stress fiber formation, as well as mislocalization and/or attenuation of sarcomeric proteins. Consistent with this, gene array and reverse transcription-PCR analyses showed down-regulation of genes encoding key cardiac transcriptional regulatory factors and proteins required for the maintenance of sarcomeric structure, function, and regulation. Chromatin immunoprecipitation analysis revealed that at least a subset of these proteins are likely regulated directly by SRF. The results presented here indicate that SRF is an essential coordinator of cardiomyocyte function due to its ability to regulate expression of numerous genes (some previously identified and at least 28 targets newly identified in this study) that are involved in multiple and disparate levels of sarcomeric function and assembly.

Animals↗
PubMed2002

Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy.

Congestive heart failure (CHF) can result from various disease states with inadequate cardiac output. CHF due to dilated cardiomyopathy (DCM) is a familial disease in 20-30% of cases and is associated with mutations in genes encoding cytoskeletal, contractile or inner-nuclear membrane proteins. We show that mutations in the gene encoding giant-muscle filament titin (TTN) cause autosomal dominant DCM linked to chromosome 2q31 (CMD1G; MIM 604145). Titin molecules extend from sarcomeric Z-discs to M-lines, provide an extensible scaffold for the contractile machinery and are crucial for myofibrillar elasticity and integrity. In a large DCM kindred, a segregating 2-bp insertion mutation in TTN exon 326 causes a frameshift, truncating A-band titin. The truncated protein of approximately 2 mD is expressed in skeletal muscle, but western blot studies with epitope-specific anti-titin antibodies suggest that the mutant protein is truncated to a 1.14-mD subfragment by site-specific cleavage. In another large family with DCM linked to CMD1G, a TTN missense mutation (Trp930Arg) is predicted to disrupt a highly conserved hydrophobic core sequence of an immunoglobulin fold located in the Z-disc-I-band transition zone. The identification of TTN mutations in individuals with CMD1G should provide further insights into the pathogenesis of familial forms of CHF and myofibrillar titin turnover.

Base Sequence↗
PubMed2002

Tibial muscular dystrophy is a titinopathy caused by mutations in TTN, the gene encoding the giant skeletal-muscle protein titin.

Tibial muscular dystrophy (TMD) is an autosomal dominant late-onset distal myopathy linked to chromosome 2q31. The linked region includes the giant TTN gene, which encodes the central sarcomeric protein, titin. We have previously shown a secondary calpain-3 defect to be associated with TMD, which further underscored that titin is the candidate. We now report the first mutations in TTN to cause a human skeletal-muscle disease, TMD. In Mex6, the last exon of TTN, a unique 11-bp deletion/insertion mutation, changing four amino acid residues, completely cosegregated with all tested 81 Finnish patients with TMD in 12 unrelated families. The mutation was not found in 216 Finnish control samples. In a French family with TMD, a Leu-->Pro mutation at position 293,357 in Mex6 was discovered. Mex6 is adjacent to the known calpain-3 binding site Mex5 of M-line titin. Immunohistochemical analysis using two exon-specific antibodies directed to the M-line region of titin demonstrated the specific loss of carboxy-terminal titin epitopes in the TMD muscle samples that we studied, thus implicating a functional defect of the M-line titin in the genesis of the TMD disease phenotype.

Amino Acid Sequence↗
PubMed1993

Development of malignant fibrous histiocytoma induced by 7,12-dimethylbenz[a]anthracene in the rat: characterization of early atypical cells.

Morphologically atypical cells were first detected in the adjacent connective tissue 98 days after implanting a paraffin pill containing 2 mg of 7,12-dimethylbenz[a]anthracene (DMBA) into the subcutaneous tissues of rats. These cells subsequently formed groups and finally produced gross malignant fibrous histiocytomas (MFH). Early atypical cells were located between proliferating fibroblasts and histiocytes in the center of a fibrous capsule surrounding the DMBA pill. They exhibited a smooth cell surface, dilated rough endoplasmic reticulum, multiple Golgi complexes, and were often associated with newly formed collagen. These cells incorporated [3H]thymidine and [3H]proline intensively, and showed weak acid phosphatase activity but no features diagnostic of macrophages (microvilli, numerous lysosomes, high acid phosphatase and non-specific esterase activities, antigens recognized by monoclonal antibodies ED1 and OX-42 and vital staining with trypan blue). There was no evidence that atypical cells differentiated into muscle cells (no expression of desmin or the alpha-sarcomeric form of actin) or Schwann cells (no expression of S-100 protein). No point mutation in the neu gene at nucleotide 2007, specific for N-ethyl-N-nitrosourea- and DMBA-induced malignant rat schwannomas, was detected by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analyses. These results support the view that malignant fibrous histiocytoma is derived from immature fibroblasts exhibiting pronounced phenotypic diversity during the later stages of carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1998

The isoenzyme-diagnostic regions of muscle-type creatine kinase, the M-260 and M-300 box, are not responsible for its binding to the myofibrillar M-band.

Muscle-type creatine kinase is known for its unique interaction with the myofibrillar M-band, but the molecular origin for this structural relationship is not well understood. A systematic sequence comparison between the highly homologous cytosolic isoforms, muscle-type and brain-type creatine kinase, yielded two isoenzyme-specific regions in the muscle-type creatine kinases, the M-260 box (residues 258-270) and the M-300 box (residues 300-315). These particular regions were conspicuous for the specific interaction of this CK isoenzyme, but not of brain-type creatine kinase, with the sarcomeric M-band. In situ diffusion assays with fluorescently labeled native, as well as mutated muscle-type creatine kinase variants, were used to study by laser confocal microscopy their association with the M-band of chemically skinned muscle fibers. Neither a set of charge mutants of the M-260 box and/or the M-300 box nor a hybrid construct of both isoforms with the entire C-terminal region derived from the brain-type isoform showed any significant alteration in the in situ M-band-binding properties when compared to the wild-type form of muscle-type creatine kinase. This indicates that in the intact protein of muscle type creatine kinase, these C-terminal isoenzyme-specific regions are not important for M-band interaction and that the actual M-band interaction domain(s) lay mostly within the N-terminal half of the molecule. The highly conserved motives (M-260 box and M-300 box) may serve an isoenzyme-specific purpose yet to be identified.

Amino Acid Sequence↗
PubMed2004

Ozz-E3, a muscle-specific ubiquitin ligase, regulates beta-catenin degradation during myogenesis.

The identities of the ubiquitin-ligases active during myogenesis are largely unknown. Here we describe a RING-type E3 ligase complex specified by the adaptor protein, Ozz, a novel SOCS protein that is developmentally regulated and expressed exclusively in striated muscle. In mice, the absence of Ozz results in overt maturation defects of the sarcomeric apparatus. We identified beta-catenin as one of the target substrates of the Ozz-E3 in vivo. In the differentiating myofibers, Ozz-E3 regulates the levels of sarcolemma-associated beta-catenin by mediating its degradation via the proteasome. Expression of beta-catenin mutants that reduce the binding of Ozz to endogenous beta-catenin leads to Mb-beta-catenin accumulation and myofibrillogenesis defects similar to those observed in Ozz null myocytes. These findings reveal a novel mechanism of regulation of Mb-beta-catenin and the role of this pool of the protein in myofibrillogenesis, and implicate the Ozz-E3 ligase in the process of myofiber differentiation.

Aging↗
PubMed1995

Mutations in the cardiac myosin binding protein-C gene on chromosome 11 cause familial hypertrophic cardiomyopathy.

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.

Adolescent↗
PubMed1997

Effect of a dominant negative ras on myocardial hypertrophy by using adenoviral-mediated gene transfer.

BACKGROUND: The small guanosine triphosphate-binding protein ras regulates a signal transduction cascade linking cell surface receptors to mitogen-activated protein kinase (MAPK). Because the molecular signaling mechanisms underlying cardiac hypertrophy remain unclear, the current study examined the regulatory role of ras in both the biochemical and morphologic aspects of hypertrophy. METHODS: Adenoviral-mediated gene transfer was used to express a dominant negative mutant of ras (rasN17) at high efficiency in primary neonatal ventricular myocytes. Beta-galactosidase staining and Western blot analysis confirmed successful transfection and expression of the rasN17 gene product. MAPK activity was measured by an in vitro kinase assay resulting in radioactive phosphorus labeled product. Morphologic hypertrophy was assessed by fluorescein-conjugated phalloidin. RESULTS: Compared with uninfected or control adenoviral-infected cells, myocytes infected with rasN17 demonstrated attenuated basal MAPK activity. In contrast, rasN17 expression did not affect endothelin 1-induced MAPK activation. Morphologic studies showed that although rasN17 produced a phenotypic difference in the basal state, the ability of cardiac myocytes to morphologically respond to endothelin 1 stimulation, as manifested by sarcomeric reorganization, remained unaltered by the expression of the rasN17 gene product. CONCLUSIONS: Endothelin 1-stimulated MAPK activation and endothelin 1-induced morphologic hypertrophy are ras-independent processes.

Adenoviridae↗
PubMed2000

[Genotype-phenotype correlations in familial hypertrophic cardiomyopathy].

Familial hypertrophic cardiomyopathy(FHC) is a complex cardiac disease with unique pathophysiological characteristics and a great diversity of morphological, functional, and clinical features. The results of molecular genetic studies have shown that FHC is a disease of the sarcomere involving eight different genes encoding proteins of the myofibrillar apparatus. Recent advances in genetic studies have provided insights into the heterogeneity of FHC clinical features. Analyses of genotype-phenotype correlations in FHC have shown that the age-related penetrance, the pattern of the left ventricular hypertrophy, and the prognosis are different depending on the disease causing genes or the disease causing mutations. Understanding of the genotype-phenotype correlations in FHC is useful for management of the disease.

Aging↗
PubMed2000

Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles.

Flightin is a multiply phosphorylated, 20-kD myofibrillar protein found in Drosophila indirect flight muscles (IFM). Previous work suggests that flightin plays an essential, as yet undefined, role in normal sarcomere structure and contractile activity. Here we show that flightin is associated with thick filaments where it is likely to interact with the myosin rod. We have created a null mutation for flightin, fln(0), that results in loss of flight ability but has no effect on fecundity or viability. Electron microscopy comparing pupa and adult fln(0) IFM shows that sarcomeres, and thick and thin filaments in pupal IFM, are 25-30% longer than in wild type. fln(0) fibers are abnormally wavy, but sarcomere and myotendon structure in pupa are otherwise normal. Within the first 5 h of adult life and beginning of contractile activity, IFM fibers become disrupted as thick filaments and sarcomeres are variably shortened, and myofibrils are ruptured at the myotendon junction. Unusual empty pockets and granular material interrupt the filament lattice of adult fln(0) sarcomeres. Site-specific cleavage of myosin heavy chain occurs during this period. That myosin is cleaved in the absence of flightin is consistent with the immunolocalization of flightin on the thick filament and biochemical and genetic evidence suggesting it is associated with the myosin rod. Our results indicate that flightin is required for the establishment of normal thick filament length during late pupal development and thick filament stability in adult after initiation of contractile activity.

Animals↗
PubMed2005

A Caenorhabditis elegans model of the myosin heavy chain IIa E706K [corrected] mutation.

Mutations in myosin heavy chain (MyHC) genes recently have been shown to be associated with various forms of congenital myopathies: myosin myopathies. The MyHC IIa E706K mutation is associated with congenital joint contractures, early-onset muscle weakness, and progressive course with moderate to severe muscle weakness later in life. To study the pathogenicity of this MyHC mutation, we investigated the effect of the corresponding mutation (E710K) in the major MyHC isoform (MyHC B) of the body wall muscle of the nematode Caenorhabditis elegans. Worms with null mutations in the MyHC B gene (unc-54) are severely paralyzed and depleted of thick filaments in the body wall muscle sarcomeres. unc-54 null mutants with extrachromosomal arrays of a gene construct including the entire wild-type unc-54 gene were partially rescued as determined by a motility assay and by morphological analysis of the body wall muscle. Analysis of unc-54 null mutants with extrachromosomal arrays of the unc-54 gene with the E710K mutation were severely paralyzed but showed formation of thick filaments in the body wall muscle. We conclude that the MyHC E706K (E710K in C. elegans) mutation is pathogenic and that the effect is primarily functional rather than structural because thick filaments are formed. The C. elegans model may be useful to study suspected pathogenic mutations in MyHC genes associated with human muscle diseases.

Animals↗
PubMed2004

C-terminal lysines determine phospholipid interaction of sarcomeric mitochondrial creatine kinase.

High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

Amino Acid Sequence↗
PubMed2003

Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers.

Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln0, a flightin null mutant, 2) Mhc13, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc6, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln0 and Mhc13 but not in Mhc6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln0 and Mhc13 but not in Mhc6. Tension generation under isometric conditions was not impaired in fln0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.

Animals↗
PubMed2004

Desminopathies in muscle disease.

A recently identified class of myopathies is produced by abnormal desmin, and is characterized by a disorganization of the desmin filament network, the accumulation of insoluble desmin-containing aggregates, and destructive changes in the sarcomeric organization of striated muscles. The desmin filaments interact with various other cytoskeletal proteins. The distinct clinical phenotypes are heterogeneous, with progressive skeletal myopathy, cardiomyopathy, and respiratory insufficiency as the most prominent features. Most of the desmin mutations are autosomal dominant. Identification of the causal genetic mutations shows that the desmin gene is not the only gene implicated in desminopathies; other genes encoding desmin-associated proteins, such as alpha-B-crystallin, and synemin may also be involved. Patients with mutations in their alpha-B-crystallin gene, which produce similar skeletal and cardiac myopathies, also have opaque lenses. Knockout mice have helped to reveal the fundamental role of desmin filaments in cell architecture, sarcomere alignment, myofibril organization, and the distribution of mitochondria. Transgenic mice, which accumulate aggregates of desmin and associated proteins in their muscles, show that the loss of desmin intermediate function as a result of mutations in desmin itself, or in the desmin-associated constituents, is important for disease progression.

Animals↗
PubMed2006

Zasp/Cypher internal ZM-motif containing fragments are sufficient to co-localize with alpha-actinin--analysis of patient mutations.

Z-band alternatively spliced PDZ-containing protein (ZASP/Cypher) has an important role in maintaining Z-disc stability in striated and cardiac muscle. ZASP/Cypher interacts through its PDZ domain with the major Z-disc actin cross-linker, alpha-actinin. ZASP/Cypher also has a conserved sequence called the ZM-motif, and it is found in two alternatively spliced exons 4 and 6. We have shown earlier that the ZM-motif containing internal regions of two related proteins ALP and CLP36 interact with alpha-actinin rod region, and that the ZM-motif is important in targeting ALP to the alpha-actinin containing structures in cell. Here, we show that the ZASP/Cypher internal fragments containing either ZM exon 4 or 6 co-localized with alpha-actinin in cultured myoblasts and nonmuscle cells. Fragments of 130 residues around the ZM-consensus were sufficient for localization, which is similar to our previous results of ALP. Moreover, ZASP/Cypher protein interacted directly with the alpha-actinin rod and competed with ALP in binding to the rod. During the inhibition of stress fiber assembly ZASP/Cypher and alpha-actinin co-localization could be partially disturbed, suggesting that ZASP/Cypher is bound to alpha-actinin mainly when alpha-actinin is localizing in stress fibers. Many point mutations found in cardiomyopathy patients are located in the internal region of ZASP/Cypher. However, we found no evidence that human patient mutations in the internal domain would affect the ZASP/Cypher co-localization with alpha-actinin, or that the mutations would destabilize the ZASP/Cypher protein.

Actinin↗
PubMedDate not supplied

[The early stages of the morphogenesis and tissue lineage of an experimental malignant fibrous histiocytoma].

Morphologically atypical cells were first detected on the 98th day after subcutaneous implantation to rats of a paraffin pellet containing 2 mg of 7,12-dimethylbenz(a)anthracene (DMBA). These cells subsequently formed groups and finally gave rise to malignant fibrous histiocytomas. Early atypical cells were located between proliferating fibroblasts and histiocytes in the center of a fibrous capsule surrounding the DMBA-pill. They exhibited a smooth cell surface, dilated rough endoplasmic reticulum, multiple Golgi complexes, and were often associated with newly formed collagen. These cells incorporated 3H-thymidine and 3H-proline intensively, and showed weak acid phosphatase activity, but no features typical for macrophages (microvilli, numerous lysosomes, high activity of acid phosphatase, nonspecific esterases, antigens recognized by monoclonal antibodies ED1 and OX-42, vital staining with trypan blue). Atypical cells also did not differentiate into muscle cells (no expression of desmin and the alpha-sarcomeric form of actin), nor into Schwann cells (no expression of S-100 protein). No point mutation of the neu gene at nucleotide 2007, which is specific for N-ethyl-N-nitrosourea and DMBA-induced malignant rat schwannoma cells, was detected by polymerase chain reaction-restriction fragment length polymorphism analyses of microscopically selected regions of individual 7 micron cryostat sections. These results support the view that malignant fibrous histiocytoma is derived from immature fibroblasts exhibiting pronounced phenotypic diversity during later stages of carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗