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PubMed2004

Tcap gene mutations in hypertrophic cardiomyopathy and dilated cardiomyopathy.

OBJECTIVES: We sought to explore the relationship between a Tcap gene (TCAP) abnormality and cardiomyopathy. BACKGROUND: Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) cause severe heart failure and sudden death. Recent genetic investigations have revealed that mutations of genes encoding Z-disc components, including titin and muscle LIM protein (MLP), are the primary cause of both HCM and DCM. The Z-disc plays a role in establishing the mechanical coupling of sarcomeric contraction and stretching, with the titin/Tcap/MLP complex serving as a mechanical stretch sensor. Tcap interacts with the calsarcin, which tethers the calcineurin to the Z-disc. METHODS: The TCAP was analyzed in 346 patients with HCM (236 familial and 110 sporadic cases) and 136 patients with DCM (34 familial and 102 sporadic cases). Two different in vitro qualitative assays-yeast two-hybrid and glutathion S-transferase pull-down competition-were performed in order to investigate functional changes in Tcap's interaction with MLP, titin, and calsarcin-1 caused by the identified mutations and a reported DCM-associated mutation, R87Q. RESULTS: Two TCAP mutations, T137I and R153H, were found in patients with HCM, and another TCAP mutation, E132Q, was identified in a patient with DCM. It was demonstrated by the qualitative assays that the HCM-associated mutations augment the ability of Tcap to interact with titin and calsarcin-1, whereas the DCM-associated mutations impair the interaction of Tcap with MLP, titin, and calsarcin-1. CONCLUSIONS: These observations suggest that the difference in clinical phenotype (HCM or DCM) may be correlated with the property of altered binding among the Z-disc components.

Asian People↗
PubMed2000

Physiological consequences of tropomyosin mutations associated with cardiac and skeletal myopathies.

Mutations have been identified in alpha-tropomyosin (Tm), a key regulatory protein in striated muscle cells, that are associated with a human cardiac myopathy, hypertrophic cardiomyopathy (FHC) and a human skeletal myopathy, nemaline myopathy (NM). In this review, we highlight experiments aimed at identifying the underlying mechanisms by which mutations in alpha-Tm cause inherited diseases of cardiac and skeletal muscle. Gene transfer of normal and mutant alpha-Tm to isolated adult cardiac myocytes was used to study the primary effects of mutant alpha-Tm proteins on the structure and contractile function of fully differentiated striated muscle cells. Both FHC and NM mutant alpha-Tm proteins incorporated normally into the adult muscle sarcomere, similar to normal Tm but exerted differential "dominant-negative" effects on the contractile function of the muscle cell. FHC mutant alpha-Tm proteins produced hypersensitivity of Ca2+-activated force production with a hierarchy that was related to the clinical severity of each mutation. Conversely, the NM mutant alpha-Tm produced a hyposensitivity of Ca2+-activated force production that may underlie, at least in part, the muscle weakness observed in NM. Taken together, the results suggest that the differential changes in the ability of the mutant Tm proteins to regulate muscle contraction in response to changing Ca2+ concentrations underlie the differential clinical presentation of the cardiac and skeletal muscle myopathies associated with mutations in alpha-Tm.

Amino Acid Sequence↗
PubMed1993

mRNA surveillance by the Caenorhabditis elegans smg genes.

mRNAs that contain premature stop codons are unstable in most eukaryotes, but the mechanism of their degradation is largely unknown. We demonstrate that functions of the six C. elegans smg genes are necessary for rapid turnover of nonsense mutant mRNAs of the unc-54 myosin heavy chain gene. Nonsense alleles of unc-54 express mRNAs that are unstable in smg(+) genetic backgrounds but have normal or near normal stability in smg(-) backgrounds. smg mutations also stabilize mRNA of unc-54(r293), a small deletion that removes the unc-54 polyadenylation site and expresses an aberrant mRNA. Most unc-54 nonsense mutations are recessive in both smg(+) and smg(-) genetic backgrounds. However, four specific alleles are recessive when smg(+) and dominant when smg(-). These smg-dependent dominant alleles express nonsense mutant polypeptides that disrupt thick filament and/or sarcomere assembly. All four alleles are predicted to express nonsense fragment polypeptides that contain most of the myosin globular head domain without an attached rod segment. By degrading messages that contain premature stop codons, the smg genes eliminate mRNAs that encode potentially toxic protein fragments. We propose that this system of mRNA turnover protects cells from their own errors of transcription, mRNA processing, or mRNA transport.

Animals↗
PubMed2001

Manipulating the contractile apparatus: genetically defined animal models of cardiovascular disease.

Within the last 10 years via gene targeting and transgenesis, numerous models of cardiovascular disease have been established and used to determine if a protein's presence or absence causes cardiovascular disease. By affecting the heart's protein complement in a defined manner, the function of the different mutated proteins or protein isoforms present in the contractile apparatus can be determined and pathogenic mechanism(s) explored. We can now remodel the cardiac protein profile and effect replacement of even the most abundant contractile proteins. Precise genetic manipulation allows exploration of the structure-function relationships which underlie cardiac function, and the consequences of defined mutations at the molecular, biochemical, cytological and physiologic levels can be determined.

Animals↗
PubMed2000

The UNC-112 gene in Caenorhabditis elegans encodes a novel component of cell-matrix adhesion structures required for integrin localization in the muscle cell membrane.

Embryos homozygous for mutations in the unc-52, pat-2, pat-3, and unc-112 genes of C. elegans exhibit a similar Pat phenotype. Myosin and actin are not organized into sarcomeres in the body wall muscle cells of these mutants, and dense body and M-line components fail to assemble. The unc-52 (perlecan), pat-2 (alpha-integrin), and pat-3 (beta-integrin) genes encode ECM or transmembrane proteins found at the cell-matrix adhesion sites of both dense bodies and M-lines. This study describes the identification of the unc-112 gene product, a novel, membrane-associated, intracellular protein that colocalizes with integrin at cell-matrix adhesion complexes. The 720-amino acid UNC-112 protein is homologous to Mig-2, a human protein of unknown function. These two proteins share a region of homology with talin and members of the FERM superfamily of proteins. We have determined that a functional UNC-112::GFP fusion protein colocalizes with PAT-3/beta-integrin in both adult and embryonic body wall muscle. We also have determined that UNC-112 is required to organize PAT-3/beta-integrin after it is integrated into the basal cell membrane, but is not required to organize UNC-52/perlecan in the basement membrane, nor for DEB-1/vinculin to localize with PAT-3/beta-integrin. Furthermore, UNC-112 requires the presence of UNC-52/perlecan and PAT-3/beta-integrin, but not DEB-1/vinculin to become localized to the muscle cell membrane.

Animals↗
PubMed2006

Regulation of the M-cadherin-beta-catenin complex by calpain 3 during terminal stages of myogenic differentiation.

The cysteine protease calpain 3 (CAPN3) is essential for normal muscle function, since mutations in CAPN3 cause limb girdle muscular dystrophy type 2A. Previously, we showed that myoblasts isolated from CAPN3 knockout (C3KO) mice were able to fuse to myotubes; however, sarcomere formation was disrupted. In this study we further characterized morphological and biochemical features of C3KO myotubes in order to elucidate a role for CAPN3 during myogenesis. We showed that cell cycle withdrawal occurred normally in C3KO cultures, but C3KO myotubes have an increased number of myonuclei per myotube. We found that CAPN3 acts during myogenesis to specifically control levels of membrane-associated but not cytoplasmic beta-catenin and M-cadherin. CAPN3 was able to cleave both proteins, and in the absence of CAPN3, M-cadherin and beta-catenin abnormally accumulated at the membranes of myotubes. Given the role of M-cadherin in myoblast fusion, this finding suggests that the excessive myonuclear index of C3KO myotubes was due to enhanced fusion. Postfusion events, such as beta1D integrin expression and myofibrillogenesis, were suppressed in C3KO myotubes. These data suggest that the persistence of fusion observed in C3KO cells inhibits subsequent steps of differentiation, such as integrin complex rearrangements and sarcomere assembly.

Animals↗
PubMed1984

Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention.

Duchenne muscular dystrophy (DMD) is the most common sex linked lethal disease in man (one case in about 4000 male live births). The patients are wheelchair bound around the age of 8-10 years and usually die before the age of 20 years. The mutation rate, estimated by different methods and from different population studies, is in the order of 7 X 10(-5), which is higher than for any other X-linked genetic disease. Moreover, unlike other X linked diseases such as hemophilia A or Lesh-Nyhan's disease, there seems to be no sex difference for the mutation rates in DMD. Several observations of DMD in girls bearing X-autosomal translocations and linkage studies on two X chromosomal DNA restriction fragment length polymorphisms indicate that the DMD locus is situated on the short arm of the X chromosome, between Xp11 and Xp22. It may be of considerable length, and perhaps consisting of actively coding and non-active intervening DNA sequences. Thus unequal crossing over during meiosis in females could theoretically account for a considerable proportion of new mutations. However, there is no structurally or functionally abnormal protein known that might represent the primary gene product, nor has any pathogenetic mechanism leading to the observed biochemical and histological alterations been elucidated. Among the numerous pathogenetic concepts the hypothesis of a structural or/and functional defect of the muscular plasma membrane is still the most attractive. It would explain both the excess of muscular constituents found in serum of patients and carriers, such as creatine kinase (CK), as well as the excessive calcium uptake by dystrophic muscle fibres, which, prior to necrosis, could lead to hypercontraction, rupture of myofilaments in adjacent sarcomeres and by excessive Ca uptake to mitochondrial damage causing crucial energy loss. The results of studies on structural and functional membrane abnormalities in cells other than muscle tissue, e.g., erythrocytes, lymphocytes and cultured fibroblasts, indicate that the DMD mutation is probably demonstrable in these tissues. However, most of the findings are still difficult to reproduce or even controversial. DMD is an incurable disease; therefore most effort, in research as well as in practical medicine, is concentrated upon its prevention.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed2005

Possible functions of p94 in connectin-mediated signaling pathways in skeletal muscle cells.

Calpains are intracellular Ca2+ -requiring 'modulator proteases', which modulate cellular functions by limited and specific proteolysis. p94/calpain3, a skeletal-muscle specific calpain, has been one of the representative calpain species which indicates physiological importance of calpain proteolytic system; a defect of proteolytic activity of p94 causes limb girdle muscular dystrophy type2A (LGMD2A, also called 'calpainopathy'). Immunohistochemical studies on myofibrils showed that p94 localizes at the Z- and N2-line regions of sarcomeres. It was also identified by the yeast two hybrid studies that p94 binds to the N2A and M-line regions of connectin. Furthermore, genetic studies indicate that p94 is indispensable for skeletal muscles, although its precise functions are still unclear. Interestingly, connectin provides sarcomere not only with elasticity but also with binding sites to various multi-functional proteins such as muscle ankyrin repeat proteins (MARPs), muscle RING finger proteins (MURFs), titin-capping protein (T-cap/telethonin), sarcomeric-alpha-actinin, p94 etc. Binding sites for these proteins are not randomly placed along connectin but rather accumulated in the Z-, N2-, and/or M-line regions, indicating the existence of 'signal complexes' unique to each regions. The concept of these complexes are strongly supported by the facts that mutations of connectin or its binding proteins in these regions severely perturb muscle functions, as in the case of LGMD2A caused by mutations in the p94 gene. Therefore, it is hypothesized that the 'signal complexes' in the Z-, N2-, and M-lines modulate muscle cell homeostasis by transducing signals of external stimulations/stresses to trigger appropriate response at various different cellular events such as protein modification and gene expressions. In this article, we performed detailed immunohistochemical analyses of p94 on isolated single myofibers. Together with recent findings about p94, it is suggested that sarcomeric localization of p94, especially its M-line localization, is affected by the combination of cellular contexts such as contractile status of myofibrils, fiber type compositions, sarcomeric maturation, and the composition of the 'signal complexes' in each region.

Actinin↗
PubMed2005

Defining alpha-skeletal and alpha-cardiac actin expression in human heart and skeletal muscle explains the absence of cardiac involvement in ACTA1 nemaline myopathy.

Mutations in alpha-skeletal actin (ACTA1) underlie several congenital muscle disorders including nemaline myopathy (NM). Almost all ACTA1-NM patients have normal cardiac function, and, even lethally affected congenital NM patients exhibit an unremarkable gestation with decreased foetal movement just prior to birth. Although alpha-skeletal actin is thought to be the predominant sarcomeric actin in human heart (Boheler KR, Carrier L, de la Bastie D, et al. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest 1991;88:323-30 ), ACTA1-NM patients almost never exhibit a cardiac phenotype. In this study, we define the relative expression of skeletal and cardiac actin proteins in human heart and skeletal muscle. We show that alpha-cardiac actin is the predominant sarcomeric isoform in human donor hearts and in early foetal skeletal muscle development. Skeletal actin is the predominant isoform from 25 to 27 weeks gestation and is the exclusive isoform expressed in muscle from infancy through to adulthood. These findings are consistent with clinical observations of NM patients and assist us to better understand the pathogenesis of inherited myopathies and cardiomyopathies with mutations in actin.

Actins↗
PubMed1991

Functional and ultrastructural effects of a missense mutation in the indirect flight muscle-specific actin gene of Drosophila melanogaster.

A single-site mutation of the flight-muscle-specific actin gene of Drosophila melanogaster causes a substitution of glutamic acid 93 by lysine in all the actin encoded in the indirect flight muscle (IFM). In these Act88FE93K mutants, myofibrillar bundles of thick and thin filaments are present but lack Z-discs and all sarcomeric repeats. Dense filament bundles, which are probably aberrant Z-discs, are seen in myofibrils of pupal flies, but early in adult life these move to the periphery of the fibrils and are not seen in skinned adult fibres. Consistent with this observation, alpha-actinin and other high molecular weight proteins, possibly associated with Z-discs, are not detected on SDS/polyacrylamide gels or Western blots of skinned adult IFM. The mutation lies at the beginning of a loop in the small domain of actin, near the myosin binding region. However, that the mutant actin binds myosin heads is shown by (1) rigor crossbridges in electron micrographs, (2) the appropriate rise in stiffness when ATP is withdrawn in mechanical experiments, and (3) equal protection against tryptic digestion provided by rigor binding between actin and myosin in both wild-type and mutant fibres. Reversal of rigor chevron angle along some thin filaments reflects reversal of thin-filament polarity due to lattice disorder. The absence of Z-discs, alpha-actinin and two high molecular weight proteins, and binding studies by others, suggest that the substitution at residue 93 affects the binding of the mutant actin to a protein, possibly alpha-actinin, which is necessary for Z-disc assembly or maintenance.

Actins↗
PubMed2007

Abnormal distribution of calcium-handling proteins: a novel distinctive marker in core myopathies.

Central core disease (CCD) and multi-minicore disease (MmD) are muscle disorders characterized by foci of mitochondria depletion and sarcomere disorganization ("cores") in muscle fibers. Although core myopathies are the most frequent congenital myopathies, their pathogenesis remains elusive and specific diagnostic markers are lacking. Core myopathies are mostly caused by mutations in 2 sarcoplasmic reticulum proteins: the massive Ca-release channel RyR1 or the selenoprotein N (SelN) of unknown function. To search for distinctive markers and to obtain further pathophysiological insight, we identified the molecular defects in 12 core myopathy patients and analyzed the immunolocalization of 6 proteins of the Ca-release complex in their muscle biopsies. In 7 cases with RYR1 mutations (6 CCD, one MmD), RyR1 was depleted from the cores; in contrast, the other proteins of the sarcoplasmic reticulum (calsequestrin, SERCA1/2, and triadin) and the T-tubule (dihydropyridine receptor-alpha1subunit) accumulated within or around the lesions, suggesting an original modification of the Ca-release complex protein arrangement. Conversely, all Ca-related proteins were distributed normally in 5 MmD cases with SelN mutations. Our results provide an appropriate tool to orientate the differential and molecular diagnosis of core myopathies and suggest that different pathophysiological mechanisms lead to core formation in SelN- and in RyR1-related core myopathies.

Adolescent↗
PubMed1996

Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans.

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat-sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.

Alleles↗
PubMed2002

Genetic diseases of muscle.

In the last twenty years, the genetic basis for most of the inherited myopathies and muscular dystrophies has been unveiled. Diseases have been found to result from loss of function of structural components of the muscle basal lamina (e.g., MCD1A), sarcolemma (e.g., the sarcoglycanopathies), nucleus (e.g., EDMD) and sarcomere (e.g., the nemaline myopathies). A few have been associated with abnormalities in the genes for muscle enzymes (e.g., calpain and fukutin). Alternate mechanisms of pathogenesis have also recently been suggested by mutations lying outside of coding regions, such as the "field effect" of chromosomal mutations in DM2. In the future, we will likely identify the genes responsible for the remaining disorders, including many of the distal myopathies. In addition, we may also find skeletal muscle diseases associated with some of the presently non-implicated muscle proteins: syntropin, dystrobrevin, epsilon-sarcoglycan and sarcospan. The next steps may be to identify and understand the relationship of modifier genes producing the phenotypic heterogeneity of many of these diseases and to characterize those and other targets for therapeutic intervention, whether by gene therapy or by pharmacological treatment.

Genetic Markers↗
PubMed2007

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod.

cMyBP-C [cardiac (MyBP-C) myosin-binding protein-C)] is a sarcomeric protein involved both in thick filament structure and in the regulation of contractility. It is composed of eight IgI-like and three fibronectin-3-like domains (termed C0-C10). Mutations in the gene encoding cMyBP-C are a principal cause of HCM (hypertrophic cardiomyopathy). cMyBP-C binds to the LMM (light meromyosin) portion of the myosin rod via its C-terminal domain, C10. We investigated this interaction in detail to determine whether HCM mutations in beta myosin heavy chain located within the LMM portion alter the binding of cMyBP-C, and to define the precise region of LMM that binds C10 to aid in developing models of the arrangement of MyBP-C on the thick filament. In co-sedimentation experiments recombinant C10 bound full-length LMM with a K(d) of 3.52 microM and at a stoichiometry of 1.14 C10 per LMM. C10 was also shown to bind with similar affinity to LMM containing either the HCM mutations A1379T or S1776G, suggesting that these HCM mutations do not perturb C10 binding. Using a range of N-terminally truncated LMM fragments, the cMyBP-C-binding site on LMM was shown to lie between residues 1554 and 1581. Since it had been reported previously that acidic residues on myosin mediate the C10 interaction, three clusters of acidic amino acids (Glu1554/Glu1555, Glu1571/Glu1573 and Glu1578/Asp1580/Glu1581/Glu1582) were mutated in full-length LMM and the proteins tested for C10 binding. No effect of these mutations on C10 binding was however detected. We interpret our results with respect to the localization of the proposed trimeric collar on the thick filament.

Binding Sites↗
PubMed2002

Involvement of nuclear factor-kappaB and apoptosis signal-regulating kinase 1 in G-protein-coupled receptor agonist-induced cardiomyocyte hypertrophy.

BACKGROUND: Recently, reactive oxygen species (ROS) have emerged as important molecules in cardiac hypertrophy. However, the ROS-dependent signal transduction mechanism remains to be elucidated. In this study, we examined the role of an ROS-sensitive transcriptional factor, NF-kappaB, and a mitogen-activated protein kinase kinase kinase, apoptosis signal-regulating kinase 1 (ASK1), in G-protein-coupled receptor (GPCR) agonist (angiotensin II, endothelin-1, phenylephrine)-induced cardiac hypertrophy in isolated rat neonatal cardiomyocytes. METHODS AND RESULTS: Using an ROS-sensitive fluorescent dye, we observed an increase in fluorescence signal on addition of the GPCR agonists. The GPCR agonists induced NF-kappaB activation. Antioxidants such as N-acetyl cysteine, N-mercaptopropionyl glycine, and vitamin E attenuated the NF-kappaB activation. Infection of cardiomyocytes with an adenovirus expressing a degradation-resistant mutant of IkappaBalpha led to suppression of the hypertrophic responses. The GPCR agonists rapidly and transiently activated ASK1 in a dose-dependent manner. Infection of an adenovirus expressing a dominant-negative ASK1 attenuated the GPCR agonist-induced NF-kappaB activation and cardiac hypertrophy. Overexpression of a constitutively active mutant of ASK1 led to NF kappaB activation and cardiac hypertrophy. Activated ASK1-induced hypertrophy was abolished by inhibition of NF-kappaB activation. CONCLUSIONS: These data indicate that GPCR agonist-induced cardiac hypertrophy is mediated through NF-kappaB activation via the generation of ROS. ASK1 is involved in GPCR agonist-induced NF-kappaB activation and resulting hypertrophy.

Angiotensin II↗
PubMed2002

Myosin heavy chain IIa gene mutation E706K is pathogenic and its expression increases with age.

BACKGROUND: The authors recently described a new autosomal dominant myopathy (OMIM 605637 inclusion body myopathy 3) associated with a missense mutation in the myosin heavy chain (MyHC) IIa gene (MyHC IIa, Human Gene Map [HGM] locus MYH2). Young patients showed minor changes in their muscle biopsies, although dystrophic alterations and rimmed vacuoles with 15- to 20-nm tubulofilaments identical to those in sporadic inclusion body myositis (s-IBM) were observed in some of the adult (especially older) patients. The current study was undertaken to investigate the relation between expression of the mutant MyHC IIa and pathologic changes in muscle. METHODS: The expression of MyHC IIa in nine muscle specimens from six individuals carrying the mutation was analyzed by immunohistochemistry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a new reverse transcriptase--PCR method to measure the relative abundance of the various MyHC transcripts. RESULTS: Young patients with muscle weakness and minor pathologic changes in muscle expressed MyHC IIa at undetectable levels. MyHC IIa was expressed at high levels in adults with a progressive clinical course and dystrophic muscle changes. In these cases, a large number of muscle fibers were hybrids with expression of more than one MyHC isoform. Both MyHC IIa alleles were equally expressed. The relative level of MyHC IIa transcripts exceeded that of the corresponding protein, indicating an increased turnover of mutated protein. MyHC IIa expression was a consistent finding in muscle fibers with rimmed vacuoles. CONCLUSIONS: The clear correlation between pathologic changes and expression of MyHC IIa indicates that defects in MyHC may lead not only to muscle weakness but also to muscle degeneration. The consistent expression of MyHC IIa in muscle fibers with rimmed vacuoles indicates that the breakdown of sarcomeric proteins is a key element in the pathogenesis of rimmed vacuoles of s-IBM type.

Adult↗
PubMed1999

Complex patterns of alternative splicing mediate the spatial and temporal distribution of perlecan/UNC-52 in Caenorhabditis elegans.

The unc-52 gene encodes the nematode homologue of mammalian perlecan, the major heparan sulfate proteoglycan of the extracellular matrix. This is a large complex protein with regions similar to low-density lipoprotein receptors, laminin, and neural cell adhesion molecules (NCAMs). In this study, we extend our earlier work and demonstrate that a number of complex isoforms of this protein are expressed through alternative splicing. We identified three major classes of perlecan isoforms: a short form lacking the NCAM region and the C-terminal agrin-like region; a medium form containing the NCAM region, but still lacking the agrin-like region; and a newly identified long form that contains all five domains present in mammalian perlecan. Using region-specific antibodies and unc-52 mutants, we reveal a complex spatial and temporal expression pattern for these UNC-52 isoforms. As well, using a series of mutations affecting different regions and thus different isoforms of UNC-52, we demonstrate that the medium NCAM-containing isoforms are sufficient for myofilament lattice assembly in developing nematode body-wall muscle. Neither short isoforms nor isoforms containing the C-terminal agrin-like region are essential for sarcomere assembly or muscle cell attachment, and their role in development remains unclear.

Agrin↗
PubMed1999

Cardiac troponin T mutations result in allele-specific phenotypes in a mouse model for hypertrophic cardiomyopathy.

Multiple mutations in cardiac troponin T (cTnT) can cause familial hypertrophic cardiomyopathy (FHC). Patients with cTnT mutations generally exhibit mild or no ventricular hypertrophy, yet demonstrate a high frequency of early sudden death. To understand the functional basis of these phenotypes, we created transgenic mouse lines expressing 30%, 67%, and 92% of their total cTnT as a missense (R92Q) allele analogous to one found in FHC. Similar to a mouse FHC model expressing a truncated cTnT protein, the left ventricles of all R92Q lines are smaller than those of wild-type. In striking contrast to truncation mice, however, the R92Q hearts demonstrate significant induction of atrial natriuretic factor and beta-myosin heavy chain transcripts, interstitial fibrosis, and mitochondrial pathology. Isolated cardiac myocytes from R92Q mice have increased basal sarcomeric activation, impaired relaxation, and shorter sarcomere lengths. Isolated working heart data are consistent, showing hypercontractility and diastolic dysfunction, both of which are common findings in patients with FHC. These mice represent the first disease model to exhibit hypercontractility, as well as a unique model system for exploring the cellular pathogenesis of FHC. The distinct phenotypes of mice with different TnT alleles suggest that the clinical heterogeneity of FHC is at least partially due to allele-specific mechanisms.

Alleles↗